DNA encoding a human 5-HT 1F receptor and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid molecule encoding a human 5-HT 1F  receptor, an isolated protein which is a human 5-HT 1F  receptor, vectors comprising an isolated nucleic acid molecule encoding a human 5-HT 1F  receptors, mammalian cells comprising such vectors, antibodies directed to the human 5-HT 1F  receptor, nucleic acid probes useful for detecting nucleic acid encoding human 5-HT 1F  receptors, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a human 5-HT 1F  receptor, pharmaceutical compounds related to human 5-HT 1F  receptors, and nonhuman transgenic animals which express DNA a normal or a mutant human 5-HT 1F  receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatment involving the human 5-HT 1F  receptor.

This is a division of application Ser. No. 07/817,920, filed Jan. 8,1992, issued Nov. 1, 1994 as U.S. Pat. No. 5,360,735.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced bypartial citations within parentheses. The disclosures of thesepublications in their entireties are hereby incorporated by reference inthis application in order to more fully describe the state of the art towhich this invention pertains.

Since the purification of a pressor substance in blood serum termedserotonin (Rapport et al., 1947) and later identified as5-hydroxytryptamine (5-HT)(Rapport, 1949), there has been a plethora ofreports demonstrating that this indoleamine not only plays a role in thefunctioning of peripheral tissues but, indeed, performs a key role inthe brain as a neurotransmitter. Certainly, the anatomical localizationof serotonin and serotonergic neurons in both the peripheral and centralnervous systems supports its role in such diverse physiologic andbehavioral functions as pain perception, sleep, aggression, sexualactivity, hormone secretion, thermoregulation, motor activity,cardiovascular function, food intake and renal regulation (For reviewsee Green, 1985; Osborne and Hamon, 1988; Sanders-Bush, 1988; Peroutka,1991). Taken together, it appears that serotonin plays an important rolein homeostasis and in modulating responsiveness to environmentalstimuli. Accordingly, studies demonstrating that abnormalities in theserotonergic system may be associated with disease states has created adrug development effort towards agents which may selectively modulatethe function of serotonin (Glennon, 1990).

In relation to the characterization of physiologic or biochemicalresponses resulting from the release of serotonin are simultaneousinvestigations examining the receptor sites responsible for the actionselicited by the indoleamine transmitter. Following early in vitropharmacological assays describing the existence of two differentserotonin receptors, designated as D and M, in the guinea pig ileum(Gaddum and Picarelli, 1957), the advent of receptor binding techniquein the 1970's has brought to light during the last decade the diversityof 5-HT receptors existing in both the brain and peripheral tissues.Thus, although the concept of D and M receptors has not beeninvalidated, serotonin receptors not fitting either category have beenidentified using radioligand methods. To date using this technique,there appears to be four classes of serotonin receptors found in thebrain: 5-HT₁, 5-HT₂, 5-HT₃ and, putatively, 5-HT₄ (Peroutka, 1991).Furthermore, 5-HT₁ sites have been subclassified as: 5-HT_(1A),5-HT_(1B), 5-HT_(1C), 5-HT_(1D) (Hamon et al., 1990) and 5-HT_(1E)(Leonhardt et al., 1989). Although a detailed characterization of the5-HT_(1E) binding site is lacking, extensive pharmacologic, biochemicaland functional properties have clearly shown that the other foursubtypes of 5-HT₁ sites are receptors according to classical criteria.

During the last few years, the field of molecular biology has providedan important facet to receptor research by cloning these proteins andallowing more precise characterizations in isolated systems (Hartig etal, 1990). This has been accomplished for the 5-HT_(1A) (Fargin et al.,1988), 5-HT_(1C) (Julius et al., 1988), 5-HT_(1D) (Branchek et al.,1990) and 5-HT₂ receptors (Pritchett et al., 1988). Thus, there is nodoubt that these binding sites represent "true" functional receptors.Indeed, the pharmacological characterization of serotonin receptorsinvolved in various physiological or biochemical functions is a keycomponent of drug development for the serotonergic system. As one candeduce from the diversity of serotonin binding sites, many targets areavailable for advancement in selective drug design. The coupling ofmolecular biological methods to pharmacological characterizationparticularly for cloned human receptors will open new avenues forpharmaceutical development which has not been previously explored.

This study is a pharmacological characterization of a serotonergicreceptor clone with a binding profile different from that of anyserotonergic receptor to date. In keeping with the nomenclaturepresently accepted for serotonin receptors, this novel site will betermed a 5-HT_(1F) receptor based upon the fact that it possesses highaffinity for the endogenous neurotransmitter, 5-HT.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid molecule encoding ahuman 5-HT_(1F) receptor (Seq. I.D. No. 1).

This invention also provides an isolated protein which is a human5-HT_(1F) receptor (Seq. I.D. Nos. 2, 7).

This invention provides a vector comprising an isolated nucleic acidmolecule encoding a human 5-HT_(1F) receptor.

This invention also provides vectors such as plasmids comprising a DNAmolecule encoding a human 5-HT_(1F) receptor, adapted for expression ina bacterial cell, a yeast cell, or a mammalian cell which additionallycomprise the regulatory elements necessary for expression of the DNA inthe bacterial, yeast, or mammalian cells so located relative to the DNAencoding the 5-HT_(1F) receptor as to permit expression thereof.

This invention provides a mammalian cell comprising a DNA moleculeencoding a human 5-HT_(1F) receptor.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a human 5-HT_(1F) receptor can bind toa human 5-HT_(1F) receptor which comprises contacting a mammalian cellcomprising an isolated DNA molecule encoding a human 5-HT_(1F) receptorwith the ligand under conditions permitting binding of ligands known tobind to a 5-HT_(1F) receptor, detecting the presence of any of theligand bound to a human 5-HT_(1F) receptor, and thereby determiningwhether the ligand binds to a human 5-HT_(1F) receptor.

This invention also provides a method for determining whether a ligandnot known to be capable of binding to the human 5-HT_(1F) receptor canfunctionally activate its activity or prevent the action of a ligandwhich does so. This comprises contacting a mammalian cell comprising anisolated DNA molecule which encodes a human 5-HT_(1F) receptor with theligand under conditions permitting the activation or blockade of afunctional response, detected by means of a bioassay from the mammaliancell such as a second messenger response, and thereby determiningwhether the ligand activates or prevents the activation of the human5-HT_(1F) receptor functional output.

This invention further provides a method of screening drugs to identifydrugs which specifically interact with, and bind to, the human 5-HT_(1F)receptor on the surface of a cell which comprises contacting a mammaliancell comprising an isolated DNA molecule encoding a human 5-HT_(1F)receptor with a plurality of drugs, determining those drugs which bindto the mammalian cell, and thereby identifying drugs which specificallyinteract with, and bind to, a human 5-HT_(1F) receptor.

This invention also provides a method of screening drugs to identifydrugs which interact with, and activate or block the activation of, thehuman 5-HT_(1F) receptor on the surface of a cell which comprisescontacting the mammalian cell comprising an isolated DNA moleculeencoding and expressing a human 5-HT_(1F) receptor with a plurality ofdrugs, determining those drugs which activate or block the activation ofthe receptor in the mammalian cell using a bioassay such as a secondmessenger assays, and thereby identifying drugs which specificallyinteract with, and activate or block the activation of, a human5-HT_(1F) receptor.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human 5-HT_(1F) receptor.

This invention also provides a method of detecting expression of the5-HT_(1F) receptor on the surface of a cell by detecting the presence ofmRNA coding for a 5-HT_(1F) receptor which comprises obtaining totalmRNA from the cell and contacting the mRNA so obtained with a nucleicacid probe comprising a nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence included within thesequence of a nucleic acid molecule encoding a human 5-HT_(1F) receptorunder hybridizing conditions, detecting the presence of mRNA hybridizedto the probe, and thereby detecting the expression of the 5-HT_(1F)receptor by the cell.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a human 5-HT_(1F) receptor so as to prevent translation ofthe mRNA molecule.

This invention provides an antibody directed to a human 5-HT_(1F)receptor.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a human 5-HT_(1F) receptor. This invention also provides atransgenic nonhuman mammal expressing DNA encoding a human 5-HT_(1F)receptor so mutated as to be incapable of normal receptor activity, andnot expressing native 5-HT_(1F) receptor. This invention furtherprovides a transgenic nonhuman mammal whose genome comprises antisenseDNA complementary to DNA encoding a human 5-HT_(1F) receptor so placedas to be transcribed into antisense mRNA which is complementary to mRNAencoding a 5-HT_(1F) receptor and which hybridizes to mRNA encoding a5-HT_(1F) receptor thereby reducing its translation.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1F) receptors whichcomprises producing a transgenic nonhuman animal whose levels of human5-HT_(1F) receptor expression are varied by use of an inducible promoterwhich regulates human 5-HT_(1F) receptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1F) receptors whichcomprises producing a panel of transgenic nonhuman animals eachexpressing a different amount of human 5-HT_(1F) receptor.

This invention provides a method for diagnosing a predisposition to adisorder associated with the expression of a specific human 5-HT_(1F)receptor allele which comprises: a. obtaining DNA of subjects sufferingfrom the disorder; b. performing a restriction digest of the DNA with apanel of restriction enzymes; c. electrophoretically separating theresulting DNA fragments on a sizing gel; d. contacting the resulting gelwith a nucleic acid probe capable of specifically hybridizing to DNAencoding a human 5-HT_(1F) receptor and labelled with a detectablemarker; e. detecting labelled bands which have hybridized to the DNAencoding a human 5-HT_(1F) receptor labelled with a detectable marker tocreate a unique band pattern specific to the DNA of subjects sufferingfrom the disorder; f. preparing DNA obtained for diagnosis by steps a-e;and g. comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.

This invention provides a method of preparing the isolated 5-HT_(1F)receptor which comprises inducing cells to express 5-HT_(1F) receptor,recovering the receptor from the resulting cells and purifying thereceptor so recovered.

This invention also provides a method of preparing the isolated5-HT_(1F) receptor which comprises inserting nucleic acid encoding5-HT_(1F) receptor in a suitable vector, inserting the resulting vectorin a suitable host cell, recovering the receptor produced by theresulting cell, and purifying the receptor so recovered.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a receptor so as to prevent translation of the mRNAmolecule.

This invention also provides a transgenic nonhuman mammal expressing DNAencoding a receptor.

This invention further provides a transgenic nonhuman mammal expressingDNA encoding a receptor so mutated as to be incapable of normal receptoractivity, and not expressing native receptor.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a transgenic nonhuman animal whose levels of receptorexpression are varied by use of an inducible promoter which regulatesreceptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a panel of transgenic nonhuman animals each expressing adifferent amount of the receptor.

This invention further provides a transgenic nonhuman mammal whosegenome comprises antisense DNA complementary to DNA encoding a receptorso placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the receptor and which hybridizes to mRNAencoding the receptor thereby preventing its translation.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a receptor can bind to a receptorwhich comprises contacting a mammalian cell comprising an isolated DNAmolecule encoding the receptor with the ligand under conditionspermitting binding of ligands known to bind to a receptor, detecting thepresence of any of the ligand bound to the receptor, and therebydetermining whether the ligand binds to the receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Nucleotide and deduced amino acid sequence of gene5-HT_(1F) (Seq. I.D. Nos. 1, 2, and 7).

Numbers above the nucleotide sequence indicate nucleotide position. DNAsequence was determined by the chain termination method of Sanger, etal., on denatured double-stranded plasmid templates using the enzymeSequenase. Deduced amino acid sequence (single letter code) of a longopen reading frame is shown.

FIGS. 2A-2D. Comparison of the human 5-HT_(1F) receptor primarystructures with other serotonin receptors (Seq. I.D. Nos.: 5-HT_(1A)--3; 5-HT_(1C) --4; 5-HT_(1D)α --5; 5-HT_(1D)β --6; 5-HT_(1F) --7; 5-HT₂--8).

Amino acid sequences (single letter code) are aligned to optimizehomology. The putative transmembrane spanning domains are indicated bystars and identified by Roman numerals (TM I-VII).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the 5-HT receptor family is defined as the group ofmammalian proteins that function as receptors for serotonin. A 5-HTreceptor subfamily is defined as a subset of proteins belonging to the5-HT receptor family which are encoded by genes which exhibit homologyof greater than 72% or higher with each other in their deduced aminoacid sequences within presumed transmembrane regions (linearlycontiguous stretches of hydrophobic amino acids, bordered by charged orpolar amino acids, that are long enough to form secondary proteinstructures that span a lipid bilayer). Four human 5-HT receptorsubfamilies can be distinguished based on the information presentlyavailable: 5-HT₁, 5-HT₂, 5-HT₃, and 5-HT₄ (Peroutka, 1991). The 5-HT₂receptor subfamily contains the human 5-HT₂ receptor. Although no otherhuman members of this family have been described, the rat 5-HT₂ receptor(Pritchett, et al. 1988; Julius, et al. Proc. Natl. Acad. Sci. U.S.A.87:928-932, 1990) and the rat 5HT_(1C) receptor (Julius, et al. 1988)constitute a rat 5-HT receptor subfamily. The 5-HT₁ subfamily has beensubdivided further as: 5-HT_(1A), 5-HT_(1B), 5-HT_(1C), 5-HT_(1D) (Hamonet al., 1990) and 5-HT_(1E) (Leonhardt et al., 1989). The 5-HT_(1A)subfamily contains the human 5-HT_(1A) receptor, also known as G-21(Fargin, et al. 1988) The 5-HT_(1D) receptor subfamily contains twomembers, the 5-HT_(1D-1) receptor (also termed 5-HT_(1D)α) and the5-HT_(1D-2) receptor (also termed 5-HT_(1D)β). The 5-HT_(1F) subfamilycontains the human 5-HT_(1F) receptor (also termed clone h116a).Although this definition differs from the pharmacological definitionused earlier, there is significant overlap between the presentdefinition and the pharmacological definition. Members of the 5-HT_(1F)receptor subfamily so described include the 5-HT_(1F) receptor and anyother receptors which have a greater than 72% homology to the DNA andamino acid sequence shown in FIGS. 1A-1C (Seq. I.D. Nos. 1, 2, and 7)according to the definition of "subfamily". This invention relates tothe discovery of the first member of the human 5-HT_(1F) receptorsubfamily.

This invention provides an isolated nucleic acid molecule encoding ahuman 5-HT_(1F) receptor (Seq. I.D. No. 1). As used herein, the term"isolated nucleic acid molecule" means a nucleic acid molecule that is,a molecule in a form which does not occur in nature. Such a receptor isby definition a member of the 5-HT_(1F) receptor subfamily. Therefore,any receptor which meets the defining criteria given above is a human5-HT_(1F) receptor. One means of isolating a human 5-HT_(1F) receptor isto probe a human genomic library with a natural or artificially designedDNA probe, using methods well known in the art. DNA probes derived fromthe human receptor gene 5-HT_(1F) are particularly useful probes forthis purpose. DNA and cDNA molecules which encode human 5-HT_(1F)receptors may be used to obtain complementary genomic DNA, cDNA or RNAfrom human, mammalian or other animal sources, or to isolate relatedcDNA or genomic clones by the screening of cDNA or genomic libraries, bymethods described in more detail below. Transcriptional regulatoryelements from the 5' untranslated region of the isolated clones, andother stability, processing, transcription, translation, and tissuespecificity-determining regions from the 3' and 5' untranslated regionsof the isolated genes are thereby obtained. Examples of a nucleic acidmolecule are an RNA, cDNA, or isolated genomic DNA molecule encoding ahuman 5-HT_(1F) receptor. Such molecules may have coding sequencessubstantially the same as the coding sequence shown in FIGS. 1A-1C. TheDNA molecule of FIG. 1 encodes the sequence of the human 5-HT_(1F)receptor gene (Seq. I.D. No. 1).

This invention further provides a cDNA molecule of encoding a human5-HT_(1F) receptor having a coding sequence substantially the same asthe coding sequence shown in FIGS. 1A-1C (Seq. I.D. No. 1). Thismolecule is obtained by the means described above.

This invention also provides an isolated protein which is a human5-HT_(1F) receptor. As used herein, the term "isolated protein means aprotein molecule free of other cellular components. An example of suchprotein is an isolated protein having substantially the same amino acidsequence as the amino acid sequence shown in FIGS. 1A-1C (Seq. I.D. Nos.2, 7) which is a human 5-HT_(1F) receptor. One means for obtainingisolated 5-HT_(1F) receptor is to express DNA encoding the receptor in asuitable host, such as a bacterial, yeast, or mammalian cell, usingmethods well known in the art, and recovering the receptor protein afterit has been expressed in such a host, again using methods well known inthe art. The receptor may also be isolated from cells which express it,in particular from cells which have been transfected with the expressionvectors described below in more detail.

This invention provides a vector comprising an isolated nucleic acidmolecule such as DNA, RNA, or cDNA encoding a human 5-HT_(1F) receptor.Examples of vectors are viruses such as bacteriophages (such as phagelambda), cosmids, plasmids (such as pUC18, available from Pharmacia,Piscataway, N.J.), and other recombination vectors. Nucleic acidmolecules are inserted into vector genomes by methods well known in theart. For example, insert and vector DNA can both be exposed to arestriction enzyme to create complementary ends on both molecules whichbase pair with each other and are then ligated together with a ligase.Alternatively, linkers can be ligated to the insert DNA which correspondto a restriction site in the vector DNA, which is then digested with therestriction enzyme which cuts at that site. Other means are alsoavailable. A specific example of such plasmids is a plasmid comprisingcDNA having a coding sequence substantially the same as the codingsequence shown in FIGS. 1A-1C and designated clone h116a.

This invention also provides vectors comprising a DNA molecule encodinga human 5-HT_(1F) receptor, adapted for expression in a bacterial cell,a yeast cell, or a mammalian cell which additionally comprise theregulatory elements necessary for expression of the DNA in thebacterial, yeast, or mammalian cells so located relative to the DNAencoding a human 5-HT_(1F) receptor as to permit expression thereof. DNAhaving coding sequences substantially the same as the coding sequenceshown in FIGS. 1A-1C may usefully be inserted into the vectors toexpress human 5-HT_(1F) receptors. Regulatory elements required forexpression include promoter sequences to bind RNA polymerase andtranscription initiation sequences for ribosome binding. For example, abacterial expression vector includes a promoter such as the lac promoterand for transcription initiation the Shine-Dalgarno sequence and thestart codon AUG (Maniatis, et al., Molecular Cloning, Cold Spring HarborLaboratory, 1982). Similarly, a eukaryotic expression vector includes aheterologous or homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Such vectors may be obtained commercially orassembled from the sequences described by methods well known in the art,for example the methods described above for constructing vectors ingeneral. Expression vectors are useful to produce cells that express thereceptor. Certain uses for such cells are described in more detailbelow.

This invention further provides a plasmid adapted for expression in abacterial, yeast, or, in particular, a mammalian cell which comprises aDNA molecule encoding a human 5-HT_(1F) receptor and the regulatoryelements necessary for expression of the DNA in the bacterial, yeast, ormammalian cell so located relative to the DNA encoding a human 5-HT_(1F)receptor as to permit expression thereof. Some plasmids adapted forexpression in a mammalian cell are pSVL (available from Pharmacia,Piscataway, N.J.), pcEXV-3 (Miller J. and Germain R. N., J. Exp. Med.164:1478 (1986)) and pMO5 (Branchek, T. et al, Mol. Pharm. 38:604-609(1990)). A specific example of such plasmid is a plasmid adapted forexpression in a mammalian cell comprising cDNA having coding sequencessubstantially the same as the coding sequence shown in FIGS. 1A-1C andthe regulatory elements necessary for expression of the DNA in themammalian cell which is designated pMO5-hl16a and deposited under ATCCAccession No. 75175. Those skilled in the art will readily appreciatethat numerous plasmids adapted for expression in a mammalian cell whichcomprise DNA of encoding human 5-HT_(1F) receptors and the regulatoryelements necessary to express such DNA in the mammalian cell may beconstructed utilizing existing plasmids and adapted as appropriate tocontain the regulatory elements necessary to express the DNA in themammalian cell. The plasmids may be constructed by the methods describedabove for expression vectors and vectors in general, and by othermethods well known in the art.

The deposit discussed supra, and the other deposits discussed herein,were made pursuant to, and in satisfaction of, the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

This invention provides a mammalian cell comprising a DNA moleculeencoding a human 5-HT_(1F) receptor, such as a mammalian cell comprisinga plasmid adapted for expression in a mammalian cell, which comprises aDNA molecule encoding a human 5-HT_(1F) receptor, the protein encodedthereby is expressed on the cell surface, and the regulatory elementsnecessary for expression of the DNA in the mammalian cell so locatedrelative to the DNA encoding a human 5-HT_(1F) receptor as to permitexpression thereof. Numerous mammalian cells may be used as hosts,including, for example, the mouse fibroblast cell NIH3T3, CHO cells,HeLa cells, Ltk⁻ cells, Y1 cells, etc. A particular example of an Ltk⁻cell is a cell designated L-5-HT_(1F) and deposited under ATCC AccessionNo. CRL 10957 and comprises the plasmid designated pMO5-hl16a. Anotherexample is the murine fibroblast cell line designated N-5-HT_(1F) anddeposited under ATCC Accession No. CRL 10956. Expression plasmids suchas that described supra may be used to transfect mammalian cells bymethods well known in the art such as calcium phosphate precipitation,or DNA encoding these 5-HT_(1F) receptors may be otherwise introducedinto mammalian cells, e.g., by microinjection, to obtain mammalian cellswhich comprise DNA, e.g., cDNA or a plasmid, encoding either human5-HT_(1F) receptor.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a human 5-HT_(1F) receptor can bind toa human 5-HT_(1F) receptor which comprises contacting a mammalian cellcomprising a DNA molecule encoding a human 5-HT_(1F) receptor, theprotein encoded thereby is expressed on the cell surface, with theligand under conditions permitting binding of ligands known to bind tothe 5-HT_(1F) receptor, detecting the presence of any of the ligandbound to the 5-HT_(1F) receptor, and thereby determining whether theligand binds to the 5-HT_(1F) receptor. This invention also provides amethod for determining whether a ligand not known to be capable ofbinding to the human 5-HT_(1F) receptor can functionally activate itsactivity or prevent the action of a ligand which does so. This comprisescontacting a mammalian cell comprising an isolated DNA molecule whichencodes a human 5-HT_(1F) receptor with the ligand under conditionspermitting the activation or blockade of a functional response, detectedby means of a bioassay from the mammalian cell such as a secondmessenger response, and thereby determining whether the ligand activatesor prevents the activation of the human 5-HT_(1F) receptor functionaloutput. The DNA in the cell may have a coding sequence substantially thesame as the coding sequence shown in FIGS. 1A-1C preferably, themammalian cell is nonneuronal in origin. An example of a nonneuronalmammalian cell is an Ltk⁻ cell, in particular the Ltk⁻ cell designatedL-5-HT_(1F). Another example of a non-neuronal mammalian cell to be usedfor functional assays is a murine fibroblast cell line, specifically theNIH3T3 cell designated N-5-HT_(1F). The preferred method for determiningwhether a ligand is capable of binding to the human 5-HT_(1F) receptorcomprises contacting a transfected nonneuronal mammalian cell (i.e. acell that does not naturally express any type of 5-HT or G-proteincoupled receptor, thus will only express such a receptor if it istransfected into the cell) expressing a 5-HT_(1F) receptor on itssurface, or contacting a membrane preparation derived from such atransfected cell, with the ligand under conditions which are known toprevail, and thus to be associated with, in vivo binding of the ligandsto a 5-HT_(1F) receptor, detecting the presence of any of the ligandbeing tested bound to the 5-HT_(1F) receptor on the surface of the cell,and thereby determining whether the ligand binds to, activates orprevents the activation of the 5-HT_(1F) receptor. This response systemis obtained by transfection of isolated DNA into a suitable host cellcontaining the desired second messenger system such as phosphoinositidehydrolysis, adenylate cyclase, guanylate cyclase or ion channels. Such ahost system is isolated from pre-existing cell lines, or can begenerated by inserting appropriate components of second messengersystems into existing cell lines. Such a transfection system provides acomplete response system for investigation or assay of the activity ofhuman 5-HT_(1F) receptors with ligands as described above. Transfectionsystems are useful as living cell cultures for competitive bindingassays between known or candidate drugs and ligands which bind to thereceptor and which are labeled by radioactive, spectroscopic or otherreagents. Membrane preparations containing the receptor isolated fromtransfected cells are also useful for these competitive binding assays.Functional assays of second messenger systems or their sequelae intransfection systems act as assays for binding affinity and efficacy inthe activation of receptor function. A transfection system constitutes a"drug discovery system" useful for the identification of natural orsynthetic compounds with potential for drug development that can befurther modified or used directly as therapeutic compounds to activateor inhibit the natural functions of the human 5-HT_(1F) receptor. Thetransfection system is also useful for determining the affinity andefficacy of known drugs at the human 5-HT_(1F) receptor sites.

This invention also provides a method of screening drugs to identifydrugs which specifically interact with, and bind to, the human 5-HT_(1F)receptor on the surface of a cell which comprises contacting a mammaliancell comprising a DNA molecule encoding a human 5-HT_(1F) receptor onthe surface of a cell with a plurality of drugs, determining those drugswhich bind to the mammalian cell, and thereby identifying drugs whichspecifically interact with, and bind to, the human 5-HT_(1F) receptor.This invention also provides a method of screening drugs to identifydrugs which interact with, and activate or block the activation of, thehuman 5-HT_(1F) receptor on the surface of a cell which comprisescontacting the mammalian cell comprising an isolated DNA moleculeencoding and expressing a human 5-HT_(1F) receptor with a plurality ofdrugs, determining those drugs which activate or block the activation ofthe receptor in the mammalian cell using a bioassay such as a secondmessenger assays, and thereby identifying drugs which specificallyinteract with, and activate or block the activation of, a human5-HT_(1F) receptor. The DNA in the cell may have a coding sequencesubstantially the same as the coding sequence shown in FIGS. 1-1C (Seq.I.D. No. 1). Preferably, the mammalian cell is nonneuronal in origin. Anexample of a nonneuronal mammalian cell is an Ltk⁻ cell, in particularthe Ltk⁻ cell designated L-5-HT_(1F). Another example of a non-neuronalmammalian cell to be used for functional assays is a murine fibroblastcell line, specifically the NIH3T3 cell designated N-5-HT_(1F). Drugcandidates are identified by choosing chemical compounds which bind withhigh affinity to the expressed 5-HT_(1F) receptor protein in transfectedcells, using radioligand binding methods well known in the art, examplesof which are shown in the binding assays described herein. Drugcandidates are also screened for selectivity by identifying compoundswhich bind with high affinity to one particular 5-HT_(1F) receptorsubtype but do not bind with high affinity to any other serotoninreceptor subtype or to any other known receptor site. Because selective,high affinity compounds interact primarily with the target 5-HT_(1F)receptor site after administration to the patient, the chances ofproducing a drug with unwanted side effects are minimized by thisapproach. This invention provides a pharmaceutical compositioncomprising a drug identified by the method described above and apharmaceutically acceptable carrier. As used herein, the term"pharmaceutically acceptable carrier" encompasses any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water, and emulsions, such as an oil/water or water/oil emulsion, andvarious types of wetting agents. Once the candidate drug has been shownto be adequately bio-available following a particular route ofadministration, for example orally or by injection (adequate therapeuticconcentrations must be maintained at the site of action for an adequateperiod to gain the desired therapeutic benefit), and has been shown tobe non-toxic and therapeutically effective in appropriate diseasemodels, the drug may be administered to patients by that route ofadministration determined to make the drug bio-available, in anappropriate solid or solution formulation, to gain the desiredtherapeutic benefit.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human 5-HT_(1F) receptor, for example with a coding sequenceincluded within the sequence shown in FIGS. 1A-1C. As used herein, thephrase "specifically hybridizing" means the ability of a nucleic acidmolecule to recognize a nucleic acid sequence complementary to its ownand to form double-helical segments through hydrogen bonding betweencomplementary base pairs. Nucleic acid probe technology is well known tothose skilled in the art who will readily appreciate that such probesmay vary greatly in length and may be labeled with a detectable label,such as a radioisotope or fluorescent dye, to facilitate detection ofthe probe. Detection of nucleic acid encoding human 5-HT_(1F) receptorsis useful as a diagnostic test for any disease process in which levelsof expression of the corresponding 5-HT_(1F) receptor is altered. DNAprobe molecules are produced by insertion of a DNA molecule whichencodes human 5-HT_(1F) receptor or fragments thereof into suitablevectors, such as plasmids or bacteriophages, followed by insertion intosuitable bacterial host cells and replication and harvesting of the DNAprobes, all using methods well known in the art. For example, the DNAmay be extracted from a cell lysate using phenol and ethanol, digestedwith restriction enzymes corresponding to the insertion sites of the DNAinto the vector (discussed above), electrophoresed, and cut out of theresulting gel. An example of such DNA molecule is shown in FIGS. 1A-1C.The probes are useful for `in situ` hybridization or in order to locatetissues which express this gene family, or for other hybridizationassays for the presence of these genes or their mRNA in variousbiological tissues. In addition, synthesized oligonucleotides (producedby a DNA synthesizer) complementary to the sequence of a DNA moleculewhich encodes human 5-HT_(1F) receptor of are useful as probes for thesegenes, for their associated mRNA, or for the isolation of related genesby homology screening of genomic or cDNA libraries, or by the use ofamplification techniques such as the Polymerase Chain Reaction.Synthesized oligonucleotides as described may also be used to determinethe cellular localization of the mRNA produced by the 5-HT_(1F) gene byin situ hybridization. An example of such an oligonucleotide is:5'-TCTCACCACTCTCCAAAAGGACTTGGCCATTCACCTCCTCCTTTG-3' (Seq. I.D. No. 9).

This invention also provides a method of detecting expression of a5-HT_(1F) receptor on the surface of a cell by detecting the presence ofmRNA coding for a 5-HT_(1F) receptor which comprises obtaining totalmRNA from the cell using methods well known in the art and contactingthe mRNA so obtained with a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human 5-HT_(1F) receptor under hybridizing conditions,detecting the presence of mRNA hybridized to the probe, and therebydetecting the expression of the 5-HT_(1F) receptor by the cell.Hybridization of probes to target nucleic acid molecules such as mRNAmolecules employs techniques well known in the art. In one possiblemeans of performing this method, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing a column which binds the poly-A tails of the mRNA molecules. ThemRNA is then exposed to radioactively labelled probe on a nitrocellulosemembrane, and the probe hybridizes to and thereby labels complementarymRNA sequences. Binding may be detected by autoradiography orscintillation counting. However, other methods for performing thesesteps are well known to those skilled in the art, and the discussionabove is merely an example.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a human 5-HT_(1F) receptor so as to prevent translation ofthe mRNA molecule. The antisense oligonucleotide may have a sequencecapable of binding specifically with any sequences of the cDNA moleculewhose sequence is shown in FIGS. 1A-1C. As used herein, the phrase"binding specifically" means the ability of a nucleic acid sequence torecognize a nucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs. A particular example of an antisense oligonucleotide is anantisense oligonucleotide comprising chemical analogues of nucleotides.

This invention also provides a pharmaceutical composition comprising anamount of the oligonucleotide described above effective to reduceexpression of a human 5-HT_(1F) receptor by passing through a cellmembrane and binding specifically with mRNA encoding a human 5-HT_(1F)receptor in the cell so as to prevent its translation and apharmaceutically acceptable hydrophobic carrier capable of passingthrough a cell membrane. The oligonucleotide may be coupled to asubstance which inactivates mRNA, such as a ribozyme. Thepharmaceutically acceptable hydrophobic carrier capable of passingthrough cell membranes may also comprise a structure which binds to areceptor specific for a selected cell type and is thereby taken up bycells of the selected cell type. The structure may be part of a proteinknown to bind a cell-type specific receptor, for example an insulinmolecule, which would target pancreatic cells. DNA molecules havingcoding sequences substantially the same as the coding sequence shown inFIGS. 1A-1C may be used as the oligonucleotides of the pharmaceuticalcomposition.

This invention also provides a method of treating abnormalities whichare alleviated by reduction of expression of a 5-HT_(1F) receptor whichcomprises administering to a subject an amount of the pharmaceuticalcomposition described above effective to reduce expression of the5-HT_(1F) receptor by the subject. This invention further provides amethod of treating an abnormal condition related to 5-HT_(1F) receptoractivity which comprises administering to a subject an amount of thepharmaceutical composition described above effective to reduceexpression of the 5-HT_(1F) receptor by the subject. Several examples ofsuch abnormal conditions are dementia, Parkinson's disease, feedingdisorders, pathological anxiety, schizophrenia, or a migraine headache.

Antisense oligonucleotide drugs inhibit translation of mRNA encodingthese receptors. Synthetic oligonucleotides, or other antisense chemicalstructures are designed to bind to mRNA encoding the 5-HT_(1F) receptorand inhibit translation of mRNA and are useful as drugs to inhibitexpression of 5-HT_(1F) receptor genes in patients. This inventionprovides a means to therapeutically alter levels of expression of human5-HT_(1F) receptors by the use of a synthetic antisense oligonucleotidedrug (SAOD) which inhibits translation of mRNA encoding these receptors.Synthetic oligonucleotides, or other antisense chemical structuresdesigned to recognize and selectively bind to mRNA, are constructed tobe complementary to portions of the nucleotide sequences shown in FIGS.1A-1C of DNA, RNA or of chemically modified, artificial nucleic acids.The SAOD is designed to be stable in the blood stream for administrationto patients by injection, or in laboratory cell culture conditions, foradministration to cells removed from the patient. The SAOD is designedto be capable of passing through cell membranes in order to enter thecytoplasm of the cell by virtue of physical and chemical properties ofthe SAOD which render it capable of passing through cell membranes (e.g.by designing small, hydrophobic SAOD chemical structures) or by virtueof specific transport systems in the cell which recognize and transportthe SAOD into the cell. In addition, the SAOD can be designed foradministration only to certain selected cell populations by targetingthe SAOD to be recognized by specific cellular uptake mechanisms whichbinds and takes up the SAOD only within certain selected cellpopulations. For example, the SAOD may be designed to bind to a receptorfound only in a certain cell type, as discussed above. The SAOD is alsodesigned to recognize and selectively bind to the target mRNA sequence,which may correspond to a sequence contained within the sequence shownin FIGS. 1A-1C by virtue of complementary base pairing to the mRNA.Finally, the SAOD is designed to inactivate the target mRNA sequence byany of three mechanisms: 1) by binding to the target mRNA and thusinducing degradation of the mRNA by intrinsic cellular mechanisms suchas RNAse I digestion, 2) by inhibiting translation of the mRNA target byinterfering with the binding of translation-regulating factors or ofribosomes, or 3) by inclusion of other chemical structures, such asribozyme sequences or reactive chemical groups, which either degrade orchemically modify the target mRNA. Synthetic antisense oligonucleotidedrugs have been shown to be capable of the properties described abovewhen directed against mRNA targets (J. S. Cohen, Trends in Pharm. Sci.10, 435 (1989); H. M. Weintraub, Sci. Am. January (1990) p. 40). Inaddition, coupling of ribozymes to antisense oligonucleotides is apromising strategy for inactivating target mRNA (N. Sarver et al.,Science 247, 1222 (1990)). An SAOD serves as an effective therapeuticagent if it is designed to be administered to a patient by injection, orif the patient's target cells are removed, treated with the SAOD in thelaboratory, and replaced in the patient. In this manner, an SAOD servesas a therapy to reduce receptor expression in particular target cells ofa patient, in any clinical condition which may benefit from reducedexpression of 5-HT_(1F) receptors.

This invention provides an antibody directed to the human 5-HT_(1F)receptor, for example a monoclonal antibody directed to an epitope of ahuman 5-HT_(1F) receptor present on the surface of a cell and having anamino acid sequence substantially the same as an amino acid sequence fora cell surface epitope of the human 5-HT_(1F) receptor included in theamino acid sequence shown in FIGS. 1A-1C (Seq. I.D. Nos. 2, 7). Aminoacid sequences may be analyzed by methods well known in the art todetermine whether they produce hydrophobic or hydrophilic regions in theproteins which they build. In the case of cell membrane proteins,hydrophobic regions are well known to form the part of the protein thatis inserted into the lipid bilayer which forms the cell membrane, whilehydrophilic regions are located on the cell surface, in an aqueousenvironment. Therefore antibodies to the hydrophilic amino acidsequences shown in FIGS. 1A-1C will bind to a surface epitope of a human5-HT_(1F) receptor, as described. Antibodies directed to human 5-HT_(1F)receptors may be serum-derived or monoclonal and are prepared usingmethods well known in the art. For example, monoclonal antibodies areprepared using hybridoma technology by fusing antibody producing B cellsfrom immunized animals with myeloma cells and selecting the resultinghybridoma cell line producing the desired antibody. Cells such as NIH3T3cells or Ltk⁻ cells may be used as immunogens to raise such an antibody.Alternatively, synthetic peptides may be prepared using commerciallyavailable machines and the amino acid sequence shown in FIGS. 1A-1C. Asa still further alternative, DNA, such as a cDNA or a fragment thereof,may be cloned and expressed and the resulting polypeptide recovered andused as an immunogen. These antibodies are useful to detect the presenceof human 5-HT_(1F) receptors encoded by the isolated DNA, or to inhibitthe function of the receptors in living animals, in humans, or inbiological tissues or fluids isolated from animals or humans.

This invention provides a pharmaceutical composition which comprises anamount of an antibody directed to the human 5-HT_(1F) receptor effectiveto block binding of naturally occurring ligands to the 5-HT_(1F)receptor, and a pharmaceutically acceptable carrier. A monoclonalantibody directed to an epitope of a human 5-HT_(1F) receptor present onthe surface of a cell and having an amino acid sequence substantiallythe same as an amino acid sequence for a cell surface epitope of thehuman 5-HT_(1F) receptor included in the amino acid sequence shown inFIGS. 1A-1C is useful for this purpose.

This invention also provides a method of treating abnormalities whichare alleviated by reduction of expression of a human 5-HT_(1F) receptorwhich comprises administering to a subject an amount of thepharmaceutical composition described above effective to block binding ofnaturally occurring ligands to the 5-HT_(1F) receptor and therebyalleviate abnormalities resulting from overexpression of a human5-HT_(1F) receptor. Binding of the antibody to the receptor prevents thereceptor from functioning, thereby neutralizing the effects ofoverexpression. The monoclonal antibodies described above are bothuseful for this purpose. This invention additionally provides a methodof treating an abnormal condition related to an excess of 5-HT_(1F)receptor activity which comprises administering to a subject an amountof the pharmaceutical composition described above effective to blockbinding of naturally occurring ligands to the 5-HT_(1F) receptor andthereby alleviate the abnormal condition. Some examples of abnormalconditions are dementia, Parkinson's disease, feeding disorders,pathological anxiety, schizophrenia, and a migraine headache.

This invention provides a method of detecting the presence of a5-HT_(1F) receptor on the surface of a cell which comprises contactingthe cell with an antibody directed to the human 5-HT_(1F) receptor,under conditions permitting binding of the antibody to the receptor,detecting the presence of the antibody bound to the cell, and therebythe presence of the human 5-HT_(1F) receptor on the surface of the cell.Such a method is useful for determining whether a given cell isdefective in expression of 5-HT_(1F) receptors on the surface of thecell. Bound antibodies are detected by methods well known in the art,for example by binding fluorescent markers to the antibodies andexamining the cell sample under a fluorescence microscope to detectfluorescence on a cell indicative of antibody binding. The monoclonalantibodies described above are useful for this purpose.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a human 5-HT_(1F) receptor. This invention also provides atransgenic nonhuman mammal expressing DNA encoding a human 5-HT_(1F)receptor so mutated as to be incapable of normal receptor activity, andnot expressing native 5-HT_(1F) receptor. This invention also provides atransgenic nonhuman mammal whose genome comprises antisense DNAcomplementary to DNA encoding a human 5-HT_(1F) receptor so placed as tobe transcribed into antisense mRNA which is complementary to mRNAencoding a 5-HT_(1F) receptor and which hybridizes to mRNA encoding a5-HT_(1F) receptor thereby reducing its translation. The DNA mayadditionally comprise an inducible promoter or additionally comprisetissue specific regulatory elements, so that expression can be induced,or restricted to specific cell types. Examples of DNA are DNA or cDNAmolecules having a coding sequence substantially the same as the codingsequence shown in FIGS. 1A-1C (Seq. I.D. No. 1). An example of atransgenic animal is a transgenic mouse. Examples of tissuespecificity-determining regions are the metallothionein promotor (Low,M. J., Lechan, R. M., Hammer, R. E. et al. Science 231:1002-1004 (1986))and the L7 promotor (Oberdick, J., Smeyne, R. J., Mann, J. R., Jackson,S. and Morgan, J. I. Science 248:223-226 (1990)).

Animal model systems which elucidate the physiological and behavioralroles of human 5-HT_(1F) receptors are produced by creating transgenicanimals in which the expression of a 5-HT_(1F) receptor is eitherincreased or decreased, or the amino acid sequence of the expressed5-HT_(1F) receptor protein is altered, by a variety of techniques.Examples of these techniques include: 1) Insertion of normal or mutantversions of DNA encoding a human 5-HT_(1F) receptor or homologous animalversions of these genes, by microinjection, retroviral infection orother means well known to those skilled in the art, into appropriatefertilized embryos in order to produce a transgenic animal (Hogan B. etal. Manipulating the Mouse Embryo, A Laboratory Manual, Cold SpringHarbor Laboratory (1986)). 2) Homologous recombination (Capecchi M. R.Science 244:1288-1292 (1989); Zimmer, A. and Gruss, P. Nature338:150-153 (1989)) of mutant or normal, human or animal versions ofthese genes with the native gene locus in transgenic animals to alterthe regulation of expression or the structure of these 5-HT_(1F)receptors. The technique of homologous recombination is well known inthe art. It replaces the native gene with the inserted gene and so isuseful for producing an animal that cannot express native receptor butdoes express, for example, an inserted mutant receptor, which hasreplaced the native receptor in the animal's genome by recombination,resulting in underexpression of the receptor. Microinjection adds genesto the genome, but does not remove them, and so is useful for producingan animal which expresses its own and added receptors, resulting inoverexpression of the receptor. One means available for producing atransgenic animal, with a mouse as an example, is as follows: Femalemice are mated, and the resulting fertilized eggs are dissected out oftheir oviducts. The eggs are stored in an appropriate medium such as M2medium (Hogan B. et al. Manipulating the Mouse Embryo, A LaboratoryManual, Cold Spring Harbor Laboratory (1986)). DNA or cDNA encoding ahuman 5-HT_(1F) receptor is purified from a vector (such as plasmidpMO5-h116a described above) by methods well known in the art. Induciblepromoters may be fused with the coding region of the DNA to provide anexperimental means to regulate expression of the trans-gene.Alternatively or in addition, tissue specific regulatory elements may befused with the coding region to permit tissue-specific expression of thetrans-gene. The DNA, in an appropriately buffered solution, is put intoa microinjection needle (which may be made from capillary tubing using apipet puller) and the egg to be injected is put in a depression slide.The needle is inserted into the pronucleus of the egg, and the DNAsolution is injected. The injected egg is then transferred into theoviduct of a pseudopregnant mouse (a mouse stimulated by the appropriatehormones to maintain pregnancy but which is not actually pregnant),where it proceeds to the uterus, implants, and develops to term. Asnoted above, microinjection is not the only method for inserting DNAinto the egg cell, and is used here only for exemplary purposes.

Since the normal action of receptor-specific drugs is to activate or toinhibit the receptor, the transgenic animal model systems describedabove are useful for testing the biological activity of drugs directedagainst these 5-HT_(1F) receptors even before such drugs becomeavailable. These animal model systems are useful for predicting orevaluating possible therapeutic applications of drugs which activate orinhibit these 5-HT_(1F) receptors by inducing or inhibiting expressionof the native or trans-gene and thus increasing or decreasing expressionof normal or mutant 5-HT_(1F) receptors in the living animal. Thus, amodel system is produced in which the biological activity of drugsdirected against these 5-HT_(1F) receptors are evaluated before suchdrugs become available. The transgenic animals which over or underproduce the 5-HT_(1F) receptor indicate by their physiological statewhether over or under production of the 5-HT_(1F) receptor istherapeutically useful. It is therefore useful to evaluate drug actionbased on the transgenic model system. One use is based on the fact thatit is well known in the art that a drug such as an antidepressant actsby blocking neurotransmitter uptake, and thereby increases the amount ofneurotransmitter in the synaptic cleft. The physiological result of thisaction is to stimulate the production of less receptor by the affectedcells, leading eventually to underexpression. Therefore, an animal whichunderexpresses receptor is useful as a test system to investigatewhether the actions of such drugs which result in under expression arein fact therapeutic. Another use is that if overexpression is found tolead to abnormalities, then a drug which down-regulates or acts as anantagonist to 5-HT_(1F) receptor is indicated as worth developing, andif a promising therapeutic application is uncovered by these animalmodel systems, activation or inhibition of the 5-HT_(1F) receptor isachieved therapeutically either by producing agonist or antagonist drugsdirected against these 5-HT_(1F) receptors or by any method whichincreases or decreases the expression of these 5-HT_(1F) receptors inman.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1F) receptors whichcomprises producing a transgenic nonhuman animal whose levels of human5-HT_(1F) receptor expression are varied by use of an inducible promoterwhich regulates human 5-HT_(1F) receptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1F) receptors whichcomprises producing a panel of transgenic nonhuman animals eachexpressing a different amount of human 5-HT_(1F) receptor. Such animalsmay be produced by introducing different amounts of DNA encoding a human5-HT_(1F) receptor into the oocytes from which the transgenic animalsare developed.

This invention also provides a method for identifying a substancecapable of alleviating abnormalities resulting from overexpression of ahuman 5-HT_(1F) receptor comprising administering the substance to atransgenic nonhuman mammal expressing at least one artificiallyintroduced DNA molecule encoding a human 5-HT_(1F) receptor anddetermining whether the substance alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofoverexpression of a human 5-HT_(1F) receptor. As used herein, the term"substance" means a compound or composition which may be natural,synthetic, or a product derived from screening. Examples of DNAmolecules are DNA or cDNA molecules having a coding sequencesubstantially the same as the coding sequence shown in FIGS. 1A-1C.

This invention provides a pharmaceutical composition comprising anamount of the substance described supra effective to alleviate theabnormalities resulting from overexpression of 5-HT_(1F) receptor and apharmaceutically acceptable carrier.

This invention further provides a method for treating the abnormalitiesresulting from overexpression of a human 5-HT_(1F) receptor whichcomprises administering to a subject an amount of the pharmaceuticalcomposition described above effective to alleviate the abnormalitiesresulting from overexpression of a human 5-HT_(1F) receptor.

This invention provides a method for identifying a substance capable ofalleviating the abnormalities resulting from underexpression of a human5-HT_(1F) receptor comprising administering the substance to thetransgenic nonhuman mammal described above which expresses onlynonfunctional human 5-HT_(1F) receptor and determining whether thesubstance alleviates the physical and behavioral abnormalities displayedby the transgenic nonhuman mammal as a result of underexpression of ahuman 5-HT_(1F) receptor.

This invention also provides a pharmaceutical composition comprising anamount of a substance effective to alleviate abnormalities resultingfrom underexpression of 5-HT_(1F) receptor and a pharmaceuticallyacceptable carrier.

This invention further provides a method for treating the abnormalitiesresulting from underexpression of a human 5-HT_(1F) receptor whichcomprises administering to a subject an amount of the pharmaceuticalcomposition described above effective to alleviate the abnormalitiesresulting from underexpression of a human 5-HT_(1F) receptor.

This invention provides a method for diagnosing a predisposition to adisorder associated with the expression of a specific human 5-HT_(1F)receptor allele which comprises: a) obtaining DNA of subjects sufferingfrom the disorder; b) performing a restriction digest of the DNA with apanel of restriction enzymes; c. electrophoretically separating theresulting DNA fragments on a sizing gel; d) contacting the resulting gelwith a nucleic acid probe capable of specifically hybridizing to DNAencoding a human 5-HT_(1F) receptor and labelled with a detectablemarker; e) detecting labelled bands which have hybridized to the DNAencoding a human 5-HT_(1F) receptor labelled with a detectable marker tocreate a unique band pattern specific to the DNA of subjects sufferingfrom the disorder; f) preparing DNA obtained for diagnosis by steps a-e;and g) comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and thereby to diagnose predisposition to the disorder if thepatterns are the same. This method may also be used to diagnose adisorder associated with the expression of a specific human 5-HT_(1F)receptor allele.

This invention provides a method of preparing the isolated 5-HT_(1F)receptor which comprises inducing cells to express 5-HT_(1F) receptor,recovering the receptor from the resulting cells, and purifying thereceptor so recovered. An example of an isolated 5-HT_(1F) receptor isan isolated protein having substantially the same amino acid sequence asthe amino acid sequence shown in FIGS. 1A-1C (Seq. I.D. Nos. 2, 7). Forexample, cells can be induced to express receptors by exposure tosubstances such as hormones. The cells can then be homogenized and thereceptor isolated from the homogenate using an affinity columncomprising, for example, serotonin or another substance which is knownto bind to the receptor. The resulting fractions can then be purified bycontacting them with an ion exchange column, and determining whichfraction contains receptor activity or binds anti-receptor antibodies.

This invention provides a method of preparing the isolated 5-HT_(1F)receptor which comprises inserting nucleic acid encoding 5-HT_(1F)receptor in a suitable vector, inserting the resulting vector in asuitable host cell, recovering the receptor produced by the resultingcell, and purifying the receptor so recovered. An example of an isolated5-HT_(1F) receptor is an isolated protein having substantially the sameamino acid sequence as the amino acid sequence shown in FIGS. 1A-1C.This method for preparing 5-HT_(1F) receptor uses recombinant DNAtechnology methods well known in the art. For example, isolated nucleicacid encoding 5-HT_(1F) receptor is inserted in a suitable vector, suchas an expression vector. A suitable host cell, such as a bacterial cell,or a eukaryotic cell such as a yeast cell, is transfected with thevector. 5-HT_(1F) receptor is isolated from the culture medium byaffinity purification or by chromatography or by other methods wellknown in the art.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a receptor so as to prevent translation of the mRNAmolecule (Seq. I.D. No. 9).

This invention also provides a transgenic nonhuman mammal expressing DNAencoding a receptor.

This invention further provides a transgenic nonhuman mammal expressingDNA encoding a receptor so mutated as to be incapable of normal receptoractivity, and not expressing native receptor.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a transgenic nonhuman animal whose levels of receptorexpression are varied by use of an inducible promoter which regulatesreceptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a panel of transgenic nonhuman animals each expressing adifferent amount of the receptor.

This invention further provides transgenic nonhuman mammal whose genomecomprises antisense DNA complementary to DNA encoding a receptor soplaced as to be transcribed into antisense mRNA which is complementaryto mRNA encoding the receptor and which hybridizes to mRNA encoding thereceptor thereby preventing its translation.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a receptor can bind to a receptorwhich comprises contacting a mammalian cell comprising an isolated DNAmolecule encoding the receptor with the ligand under conditionspermitting binding of ligands known to bind to a receptor, detecting thepresence of any of the ligand bound to the receptor, and therebydetermining whether the ligand binds to the receptor.

Applicants have identified individual receptor subtype proteins and havedescribed methods for the identification of pharmacological compoundsfor therapeutic treatments. Pharmacological compounds which are directedagainst specific receptor subtypes provide effective new therapies withminimal side effects.

This invention identifies for the first time a new receptor protein, itsamino acid sequence, and its human gene. Furthermore, this inventiondescribes a previously unrecognized group of receptors within thedefinition of a 5-HT_(1F) receptor. The information and experimentaltools provided by this discovery are useful to generate new therapeuticagents, and new therapeutic or diagnostic assays for this new receptorprotein, its associated mRNA molecule or its associated genomic DNA. Theinformation and experimental tools provided by this discovery will beuseful to generate new therapeutic agents, and new therapeutic ordiagnostic assays for this new receptor protein, its associated mRNAmolecule, or its associated genomic DNA.

Specifically, this invention relates to the first isolation of a humancDNA and genomic clone encoding a 5-HT_(1F) receptor. A new human genefor the receptor identified herein as 5-HT_(1F) has been identified andcharacterized, and a series of related cDNA and genomic clones have beenisolated. In addition, the human 5-HT_(1F) receptor has been expressedin Ltk⁻ cells and NIH3T3 cells by transfecting the cells with theplasmid pMO5-hl16a. The pharmacological binding properties of theprotein encoded have been determined, and these binding propertiesclassify this protein as a serotonin 5-HT_(1F) receptor. Mammalian celllines expressing this human 5-HT_(1F) receptor at the cell surface havebeen constructed, thus establishing the first well-defined, culturedcell lines with which to study this 5-HT_(1F) receptor.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Materials and Methods

Polymerase Chain Reaction (PCR): The third (III) and fifth (V)transmembrane domains of the following receptors were aligned and usedto synthesize a pair of "degenerate" primers: 5-HT_(1A) (Seq. I.D. No.3), 5-HT_(1C) (Seq. I.D. No. 4), 5-HT₂ (Seq. I.D. No. 8) and the5-HT_(1D)α/β (Seq. I.D. Nos. 5 and 6, respectively) receptors (patentpending). These primers hybridize to opposite strands of targetsequences to allow amplification of the region between the correspondingtransmembrane domains. That primer which was designed to anneal totransmembrane domain III is designated 3.17 and consists of a mixture of192 different 31-mers with two inosine nucleotides; the primer whichannealed to transmembrane domain V is designated 5.5 and consists of amixture of 288 different 27-mers with five inosine nucleotides. EcoRIlinkers were included at th 5' end of primer 3.17, to facilitate thesubcloning of the amplified cDNA in pBluescript (Stratagene) vectors. 5μg of poly (A+) RNA from rat brain was reverse transcribed by avianmyeloblastosis virus reverse transcriptase (AMV) including 3 μM each of3.17 and 5.5 primers. The resulting single-stranded cDNA was used in aPCR reaction under the following conditions: 94° C. for 1 minute, 50° C.for 2 minutes and 72° C. for 3 minutes for 40 cycles. Following PCR, 90μl of the reaction was phenol:chloroform extracted and precipitated; 10μl was visualized on a gel using ethidium bromide staining. Afterprecipitation the sample was treated with T4 DNA polymerase and digestedwith EcoR1 prior to separation on a 1% agarose gel. The DNA fragment wasisolated from the gel, kinased and cloned into pBluescript. Recombinantclones were analyzed by sequencing.

Cloning and Sequencing: A human lymphocyte genomic library (Stratagene)was screened using the rat S51 fragment (obtained by PCR) as a probe.The probe was labeled with ³² P by the method of random priming(Feinberg et al., 1983). Hybridization was performed at 50° C. in asolution containing 50% formamide, 10% dextran sulfate, 5× SSC (1× SSCis 0.15M sodium chloride, 0.015M sodium citrate), 1× Denhardt's (0.02%polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and200 μg/ml of sonicated salmon sperm DNA. The filters were washed at 50°C. in 0.1× SSC containing 0.1% sodium dodecyl sulfate (SDS) and exposedat -70° C. to Kodak XAR film in the presence of an intensifying screen.Lambda phage hybridizing to the probe were plaque purified and DNA wasprepared for Southern blot analysis (Southern, 1975; Maniatis et al.,1982). For subcloning and further Southern blot analysis DNA wasinserted into pUC18 (Pharmacia, Piscataway, N.J.). Nucleotide sequenceanalysis was done by the Sanger dideoxy nucleotide chain-terminationmethod (Sanger 1977) on denatured double-stranded plasmid templatesusing Sequenase (U.S. Biochemical Corp., Cleveland, Ohio).

Expression: The entire coding region of clone hl16a was cloned into theeukaryotic expression vector pcEXV-3 (Miller, 1986). Stable cell lineswere obtained by cotransfection with the plasmid pcEXV-3 (containing the5-HT_(1F) receptor gene) and the plasmid pGCcos3neo (containing theaminoglycoside transferase gene) into Ltk⁻ cells or NIH3T3 cells usingcalcium phosphate (reagents obtained from Specialty Media, Lavellette,N.J.). The cells were grown in a controlled environment (37° C., 5% CO₂)as monolayers in Dulbecco's modified Eagle medium (Gibco, Grand Island,N.Y.) containing 25 mM glucose and supplemented with 10% bovine calfserum, 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate. Stableclones were then selected for resistance to the antibiotic G-418 andharvested membranes were screened for their ability to bind [³H]serotonin.

Membrane Preparation: Membranes were prepared from transfected Ltk-cells which were grown to 100% confluency. The cells were washed twicewith phosphate-buffered saline, scraped from the culture dishes into 5ml of ice-cold phosphate-buffered saline, and centrifuged at 200×g for 5min at 4'. The pellet was resuspended in 2.5 ml of ice-cold Tris buffer(20 mM Tris -HCl, pH 7.4 at 23°. 5 mM EDTA) and homogenized by a Wheatontissue grinder. The lysate was subsequently centrifuged at 200×g for 5min at 4' to pellet large fragments which were discarded. Thesupernatant was collected and centrifuged at 40,000×g for 20 min at 4°.The pellet resulting from this centrifugation was washed once inice-cold Tris wash buffer and finally resuspended in a final buffercontaining 50 mM Tris-HCl and 0.5 mM EDTA, pH 7.4 at 23°. Membranepreparations were kept on ice and utilized within two hours for theradioligand binding assays. Protein concentrations were determined bythe method of Bradford (1976) using bovine serum albumin as thestandard.

Radioligand Binding: [³ H]5HT binding was performed using slightmodifications of the 5-HT_(1D) assay conditions reported byHerrick-Davis and Titeler (1988) with the omission of masking ligands.Radioligand binding studies were achieved at 37° C. in a total volume of250 μl of buffer (50 mM Tris, 10 mM MgCl₂, 0.2 mM EDTA, 10 μM pargyline,0.1% ascorbate, pH 7.4 at 37° C.) in 96 well microtiter plates.Saturation studies were conducted using [³ H]5-HT at 12 differentconcentrations ranging from 0.5 nM to 100 nM. Displacement studies wereperformed using 4.5-5.5 nM [³ H]5-HT. The binding profile of drugs incompetition experiments was accomplished using 10-12 concentrations ofcompound. Incubation times were 30 min for both saturation anddisplacement studies based upon initial investigations which determinedequilibrium binding conditions. Nonspecific binding was defined in thepresence of 10 μM 5-HT. Binding was initiated by the addition of 50 μlmembrane homogenates (10-20 μg). The reaction was terminated by rapidfiltration through presoaked (0.5% polyethyleneimine) filters using 48RCell Brandel Harvester (Gaithersburg, Md.). Subsequently, filters werewashed for 5 sec with ice cold buffer (50 mM Tris HCL, pH 7.4 at 4° C.),dried and placed into vials containing 2.5 ml of Readi-Safe (Beckman,Fullerton, Calif.) and radioactivity was measured using a Beckman LS5000TA liquid scintillation counter. The efficiency of counting of [³H]5HT averaged between 45-50%. Binding data was analyzed bycomputer-assisted nonlinear regression analysis (Accufit and Accucomp,Lundon Software, Chagrin Falls, Ohio). IC₅₀ values were converted toK_(i) values using the Cheng-Prusoff equation (1973). All experimentswere performed in triplicate.

Measurement of cAMP Formation

Transfected NIH3T3 cells (estimated Bmax from one point competitionstudies=488 fmol/mg of protein) were incubated in DMEM, 5 mMtheophylline, 10 mM Hepes (4-[2-Hydroxyethyl]-1-piperazineethanesulfonicacid), 10 μM pargyline, for 20 minutes at 37° C., 5% CO₂. Drugdose-effect curves were then conducted by adding 6 different finalconcentrations of drug, followed immediately by the addition offorskolin (10 μM). Subsequently, the cells were incubated for anadditional 10 minutes at 37° C., 5% CO₂. The media was aspirated and thereaction terminated by the addition of 100 mMHCl. The plates were storedat 4° C. for 15 minutes and centrifuged for 5 minutes (500×g at 4° C. )to pellet cellular debris. Aliquots of the supernatant fraction werethen stored at -20° C. prior to assessment of cAMP formation byradioimmunoassay (cAMP Radioimmunoassay kit, Advanced Magnetics,Cambridge, Mass.).

Drugs: [³ H]5-HT (specific activity=28 Ci/mmole) was obtained from NewEngland Nuclear, Boston, Mass. All other chemicals were obtained fromcommercial sources and were of the highest grade purity available.

Results

Cloning of a Novel Gene Encoding a 5HT_(1F) Receptor

Polyadenylated (poly A+) RNA prepared from rat brain was reversetranscribed and the resulting cDNAs were subjected to amplification byPCR with the use of a set of "degenerate" primers. The synthesis ofthese primers were based on sequences corresponding to the third andfifth transmembrane segments of the current set of available serotoninreceptors. The primers were designed to amplify only serotonin specificsequences. This was accomplished, particularly with the transmembranedomain V primer, which was designed to anneal at its 3' end only to thesequence "AFY(F)IP". We have determined by sequence analysis that thepresence of an alanine (A) rather than a serine (S) in the positionimmediately amino-terminal to the sequence "FY(F)IP" is an amino acidwhich can distinguish the closely related adrenergic and dopaminergicreceptor families from the serotonergic receptor family. After 30amplification cycles, agarose gel electrophoresis revealed a clearpattern of cDNA species of approximately 250 base pairs. IndividualcDNAs were cloned directly into pBluescript and subjected to sequenceanalysis. One clone, designated S51, was observed to encode a novelserotonin receptor. We then screened a human genomic placental librarywith the PCR fragment S51. Isolation of the full-length coding regionwas obtained from a genomic clone designated hl16a.

Nucleotide Sequence and Deduced Amino Acid Sequence of hl16a

DNA sequence information obtained from clone hl16a is shown in FIGS.1A-1C. An open reading frame extending from an ATG start codon atposition 1 to a stop codon at position 1098 can encode a protein 366amino acids in length, having a relative molecular mass (M_(r)) of41,660. A comparison of this protein sequence with previouslycharacterized neurotransmitter receptors indicates that hl16a encodes areceptor which is a new member of a family of molecules which span thelipid bilayer seven times and couple to guanine nucleotide regulatoryproteins (the G protein-coupled receptor family). A variety ofstructural features which are invariant in this family were presentincluding the aspartic acid residues of transmembrane regions II andIII, the DRY sequence at the end of transmembrane region III, and theconserved proline residues of transmembrane regions IV, V, VI and VII.(Hartig et al. and references therein), were present in clone hl16a. Acomparison of the transmembrane homology of hl16a to the other clonedserotonin receptors is shown if FIG. 2 exhibits the following order ofidentity: 5-HT_(1D)α (61%), 5-HT_(1D)β (59%), 5-HT_(1A) (54%), 5-HT_(1C)(44%) and 5-HT₂ (44%).

Receptor Expression in Transfected Mammalian Cells

Saturation analysis of membranes prepared from stably transfected Ltk-cells demonstrated that the receptor expressed was saturable and of highaffinity. Scatchard plot analysis by non-linear regression revealed a Kdof 9.2±0.99 nM (mean±S.E.M., n=4) and a B_(max) 4.4±0.36 picomoles/mg ofprotein (mean±S.E.M., n=4). The percent specific binding determined atthe measured Kd value for [³ H]5-HT was greater than 85% of totalbinding. Furthermore, evidence that the receptor is coupled to aG-protein was demonstrated by the ability of Gpp(NH)p, anon-hydrolyzable analog of GTP, to inhibit the specific binding of [³H]5-HT (IC₅₀ =243±115, n_(H) =0.71±0.08, I_(max) 55.6±3.2%; mean±S.E.M.,n=3). Additional data demonstrating that this coupling to a G-protein isfunctionally relevant is provided below.

Pharmacological analysis of the receptor was accomplished by testing theability of drugs from different chemical classes to displace [³ H]5-HTspecific binding (Table 1). Of the compounds investigated, 5-HTpossessed the highest affinity which according to the classificationsystem of Peroutka and Snyder (1979) makes this site a member of the5-HT₁ class. Interestingly, 5-CT possessed low affinity and, thus,discriminates this receptor from that of the 5-HT_(1D) receptor as wellas other members of this class. The one exception appears to be therecently cloned 5-HT_(1E) receptor which also has low affinity for 5-CT(Patent Application #). Various ergoline compounds also bound with highaffinity including methylergonovine and methysergide. Excluding1-napthylpiperazine (Ki=54), piperazine derivatives had low affinity.Interestingly, the rauwolfia alkaloids, rauwolscine and yohimbine, whichare alpha-2 adrenergic antagonists had fair affinity for thisserotonergic receptor. Furthermore, miscellaneous serotonergic agentsthat possess high affinity for various receptors within the serotoninfamily including ketanserin (5-HT₂), 8-OH-DPAT (5-HT_(1A)) , DOI(5-HT_(1C) /5-HT₂), spiperone (5-HT_(1A) /5-HT₂) , pindolol (5-HT_(1A)/5-HT_(1B)) and zacopride (5-HT₃) had very poor affinity. Takentogether, the pharmacological profile of the 5-HT_(1F) receptor isunique and contrasts to that of other known serotonin receptors.Accordingly, the probability of developing selective drugs for thisreceptor subtype is increased.

                  TABLE 1    ______________________________________    Ki (nM) values of various drugs for the inhibition of [.sup.3 H]5-HT    specific    binding to clonal 5-HT.sub.1F cell membranes. Binding assays were    performed with    4.5-5.5 nM of [.sup.3 H]5-HT and 10-12 different concentrations of    each inhibitory drug. Ki values were calculated from the IC.sub.50 values    using    the Cheng-Prusoff equation. Each value is the mean ± S.E.M. of 2-4    independent determinations.    COMPOUND         Ki (nM)    ______________________________________    5-HT             10.3 ± 2.0    Sumatriptan       23.0 ± 11.0    Ergonovine       31.0 ± 1.5    Methylergonovine  31.0 ± 11.0    Methysergide     34.0 ± 4.9    5-Methoxy-N,N-DMT                     37.5 ± 1.5    1-Napthylpiperazine                     54.0 ± 3.8    Yohimbine         92.0 ± 11.0    Ergotamine       171 ± 28    α-Methyl-5-HT                     184 ± 35    NAN 190          203 ± 13    Dihydroergotamine                     276 ± 49    Metergoline      341 ± 71    2-Methyl-5-HT     413 ± 5.6    Methiothepin     652 ± 41    5-CT             717 ± 71    TFMPP            1,002 ± 85    5-MT             1,166 ± 197    SCH 23390        1,492 ± 165    5-Benzoxytryptamine                     1,495 ± 893    DP-5-CT          1,613 ± 817    DOI              1,739 ± 84    8-OH-DPAT        1,772 ± 38    5-Fluorotryptamine                     1,805 ± 220    mCPP             2,020 ± 36    Tryptamine       2,409 ± 103    Quipazine        4,668 ± 814    Ritanserin       3,521 ± 86    Propanolol       8,706 ± 97    Ketanserin       >10,000    Spiperone        >10,000    Zacopride        >10,000    Pindolol         >10,000    Mesulergine      >10,000    Harmaline        >10,000    Melatonin        >10,000    ______________________________________

cAMP Assay

Additional supporting evidence that the 5-HT_(1F) receptor isfunctionally coupled to a G-protein was obtained by testing the abilityof 5-HT as well as other representative serotonergic drugs to inhibitforskolin stimulated cAMP production in NIH3T3 cells transfected withthe 5-HT1F receptor. The endogenous indoleamine, 5-HT, produced aconcentration-related decrease in forskolin-stimulated cAMP productionwith an EC50 of 7.1 ±1.3 nM (n=4). The maximum inhibition of cAMPproduction by 5-HT was 67±5.4%. Additionally, the serotonergic compounds1-napthylpiperazine and lysergol inhibited forskolin-stimulated cAMPproduction with EC50 values of 4.5±0.2 nM and 8.8±4.3 nM (n=2),respectively.

Discussion

The deduced amino acid sequence of hl16a was analyzed to uncoverrelationships between it and the other cloned serotonin receptorsequences. Although the homology within the membrane spanning domainswas greatest with the 5-HT_(1D)α receptor (FIGS. 2A-2D), the nature ofthis newly cloned receptor could not be clearly predicted. The rationalfor this ambiguity is the interpretation of the transmembrane domainhomology (approximately 60%) to the 5-HT_(1D)α and 5-HT_(1D)β receptorsubfamily. Closely related members of a "subfamily" of serotoninreceptors (i.e. "subtypes") generally share a common transmitter andalso have similar pharmacological profiles and physiological roles (forexample, 5-HT₂ and 5-HT_(1C) or 5-HT_(1D)α and 5-HT_(1D)β). Such"subtypes" display an amino acid identity of approximately 75-80% intheir transmembrane domains. Serotonin receptors which are not membersof the same "subfamily", but are members of the serotonin "family" (inwhich the receptors use the same neurotransmitter; i.e. 5-HT₂ and5-HT_(1D)α) generally show much lower transmembrane homology(approximately 45%). Such transmembrane amino acid homologies can,therefore, give insight into the relationship between receptors and beused as predictors of receptor pharmacology. According to this type ofanalysis, although the newly cloned receptor appears to be more relatedto the 5-HT_(1D) subfamily, it is likely to be in a subfamily distinctfrom all the other serotonin receptors. Interestingly, the transmembranehomology between the 5HT_(1E) and 5HT_(1F) receptors is 72%. It istherefore possible that these receptors may be "subtypes", rather thanmembers of distinct "subfamilies".

The present pharmacological evidence substantiates the existence of anovel serotonin receptor in the human brain and peripheral tissues.Comparison of the binding affinities for various drugs observed innative membranes for other known serotonergic receptors (see Hoyer,1989) to that of the 5-HT_(1F) receptor demonstrates that thepharmacological profile does not fit any known receptor to date. Thecloning of the 5-HT_(1F) site will now allow more extensiveinvestigations into the nature of this unique serotonergic receptor.

The structure-activity relationships observed in the present studysuggest that there are important requirements for high affinity bindingto the 5-HT_(1F) receptor. Substitution or removal of the 5-hydroxygroup on serotonin significantly decreases the affinity for the receptor(e.gs., tryptamine, 5-methoxytryptamine and 5-carboxyamidotryptamine).Additionally, α-methylation and 2-methylation of 5-HT lowers itsaffinity by 20 and 40 fold, respectively, for the 5-HT_(1F) site. Incontrast to these substitutions, N,N-dimethylation of the aliphatic sidechain of the indole ring increases the affinity approximately 20 fold(unpublished observations). Interestingly,5-methoxy-N,N-dimethyltryptamine which possesses both a 5-hydroxysubstitution as well as a N,N-dimethylation has an affinity much higherthan the other 5-substituted tryptamine derivatives. Basic structuralrequirements of the ergoline derivatives demonstrate that N-methylationof the indole ring does not decrease affinity as does bulkysubstitutions. Furthermore, piperazine derivatives are not bound at highaffinity.

Notably, the application of the human 5-HT_(1F) receptor clone topharmaceutical research can lead to new drug design and development. Inthis regard, it is important to point out that the affinities ofsumatriptan, methylergonovine and methysergide for this receptor suggestthat this site may be involved in the control of migraine headaches.Certainly, these compounds have had success in the clinic for thetreatment of this debilitating disorder (Sleight et al., 1990). Notably,however, it has been thought that the action of these compounds ismediated at 5-HT1D receptors for sumatriptan and 5-HT2 receptors formethysergide. Interestingly, methylergonovine may be an activemetabolite of methysergide which can be responsible for some of thetherapeutic antimigraine effects of methysergide. This novel site withaffinity for these agents would now suggest that there is oneserotonergic receptor which may be responsible for both the pathogenesisand, accordingly, the pharmacological treatment. Importantly, the agentsprescribed for migraine are not selective for any one particularserotonin receptor and, thus, the. physiological significance of drugsacting at one specific site remains controversial (Humphrey P. P. A. etal., 1990). The notion that the 5-HT_(1F) receptor is involved inmigraine may be supported by evidence demonstrating that metergolinewhich has high affinity for the 5-HT_(1D) receptor does not block theeffects of sumatriptan in the dog saphenous vein (Sumner and Humphrey,1990) inferring that this vascular model may contain the novel 5-HT_(1F)site. Furthermore, this data can support the idea that sumatriptan actsat 5-HT_(1F) receptors as an anti-migraine drug. The potential of the5-HT_(1F) receptor as a novel target for migraine where selective drugsmay be developed is an exciting possibility which needs to be explored.

Another consideration for therapeutic application of this site may berelated to the treatment of feeding disorders such as obesity, bulimeanervosa and/or anorexia nervosa. The involvement of serotonin andfeeding behavior has received much attention during the last decade. Itis now known that many of the identified and well-characterizedserotonergic receptors are capable of modulating feeding (Blundell andLawton, 1990). Notably, serotonin uptake blockers which have been usedto treat feeding disorders act nonselectively and as such haveside-effect potential (Jimerson et al., 1990). The fact that the5-HT_(1F) receptor has been cloned from both peripheral and centralsites suggests from an anatomical standpoint that it can be found instrategic locations where feeding may be altered. Although manydifferent serotonergic receptors are involved in feeding, the search forthe one site that can be exploited for selective drug development hasyet to be found. There is no doubt that interest exists in finding drugsthat interact with the serotonin system for the treatment of feedingdisorders (Cooper, 1989).

Overall, the 5-HT_(1F) receptor can be an important site stimulated bynonselectively blocking serotonin uptake as is accomplished with certainantidepressants. In regard to this, serotonin uptake blockers areeffective in treating neuropsychiatric disorders such as depression andobsessive-compulsive illness (Asberg et al., 1986; Sleight et al., 1990;Insel et al., 1985). However, these agents have side effects and, infact, the mechanism of action for these compounds are not linked to anyparticular serotonergic receptor. The possibility that agents selectivefor the 5-HT_(1F) receptor may have clinical utility as antidepressants,for example, without the side effects attributed to current treatmentmodalities can have significant implications for drug therapy.

In summary, the pharmacological profile of the cloned human 5-HT_(1F)receptor is unique and contrasts to other known serotonergic receptors.The utility of this site expressed in a cellular system and, thus,isolated for study will create excellent opportunities in drugdevelopment directed towards a novel serotonergic receptor that may havewide-range implications for drug therapy. Ultimately, indepthinvestigations into the localization of this receptor in brain andperipheral tissue will target new sites that may lead to functionalroles of the serotonergic receptor. Indeed, the potential therapeuticapplications may extend to neuropsychiatric disorders includingdepression, anxiety, schizophrenia, dementia and obsessive-compulsiveillness as well as obesity and migraine.

Additionally, the localization of the 5-HT_(1F) receptor in the spinalcord will suggest a possible role in modulation of nociceptive stimuliwhich may lead to analgesic drug development. Furthermore, the presenceof the 5-HT1F on vascular tissue may deem this site useful forcardiovascular drug applications.

References

Asberg, M., Eriksson, B., Matensson, B., Traskman-Bendz, L. and Wagner,A.: Therapeutic effects of serotonin uptake inhibitors in depression. J.Clin. Psychiat. 47:23-35, 1986.

Bradford, M.: A rapid and sensitive method for the quantification ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochem. 72:248-254, 1976.

Blundell, J. E. and Lawton, C. L.: Serotonin receptor subtypes and theorganisation of feeding behaviour: Experimental models. In: Serotonin:From cell biology to pharmacology and therapeutics. (eds. Paoletti, R.,Vanhoutte, P. M., Brunello, N. and Maggi, F. M.) Boston: Kluwer AcademicPublishers, pp 213-219, 1990.

Branchek, T., Weinshank, R. L., Macchi, M. J., Zgombick, J. M. andHartig, P. R.: Cloning and expression of a human 5-HT1D receptor. TheSecond IUPHAR Satellite Meeting on Serotonin, Basel, Switzerland, Jul.11-13, 1990, Abstract #2.

Cheng, Y. C. and Prusoff, W. H.: Relationship between the inhibitionconstant (Ki) and the concentration of inhibitor which causes 50%inhibition (IC50) of an enzyme reaction. Biochem. Pharmacol.22:3099-3108, 1973.

Cooper, S. J.: Drugs interacting with 5-HT systems show promise fortreatment of eating disorders. TIPS 10:56-57, 1989.

Fargin, A., Raymond, J. R., Lohse, M. J., Kobilka, B. K. Caron, M. G.and Lefkowitz, R. J.: The genomic clone G-21 which resembles aβ-adrenergic receptor sequence encodes the 5-HT1A receptor. Nature335:358-360, 1988.

Feinberg, A. P., and Vogelstein, B. A technique for radiolabeling DNArestriction endonuclease fragments to high specific activity. Anal.Biochem. 132:6-13, 1983.

Gaddum, J. H. and Picarelli, Z. P.: Two kinds of tryptamine receptor.Brit. J. Pharmacol. 12:323-328, 1957.

Glennon, R. A.: Serotonin receptors: Clinical implications. Neurosci.Biobehav. Rev. 14:35-47, 1990.

Green, A. R.: Neuropharmacology of serotonin. Oxford: Oxford UniversityPress, 1985.

Hamon, M., Lanfumey, L., El Mestikawy, S., Boni, C., Miquel, M.-C.,Bolanos, F., Schechter, L. and Gozlan, H.: The main features of central5-HT1 receptors. Neuropsychopharmacol. 3(5/6):349-360, 1990.

Hartig, P. R., Kao, H.-T., Macchi, M., Adham, N., Zgombick, J.,Weinshank, R. and Branchek, T.: The molecular biology of serotoninreceptors: An overview. Neuropsychopharmacol. 3(5/6):335-347, 1990.

Herrick-Davis K. and Titeler, M.: Detection and characterization of theserotonin 5-HT_(1D) receptor in rat and human brain. J. Neurochem.50:1624-1631, 1988.

Hoyer, D.: Biochemical mechanisms of 5-HT receptor-effector coupling inperipheral tissues. In: Peripheral actions of 5-HT. (ed. Fozard, J. R.)Oxford:Oxford University Press, pp 72-99, 1989.

Humphrey, P. P. A., Feniuk, W., Perren, M. J., Beresford, I. J. M.,Skingle, M. and Whalley, E. T.: Serotonin and migraine. Ann. N.Y. Acad.Sci. 600:587-600, 1990.

Insel, T. R., Mueller, E. A., Alterman, I., Linnoila, M. and Murphy, D.L.: Obsessive-compulsive disorder and serotonin: Is there a connection?Biol. Psychiat. 20:1174-1188, 1985.

Jimerson, D. C., Lesem, M. D., Hegg, A. P. and Brewerton, T. D.:Serotonin in human eating disorders. Ann. N.Y. Acad. Sci. 600:532-544,1990.

Julius, D., MacDermott, A. B., Axel, R. and Jessell, T. M.: Molecularcharacterization of a functional cDNA encoding the serotonin 1Creceptor. Science 241:558-564, 1988.

Leonhardt, S., Herrick-Davis, K. and Titeler, M.: Detection of a novelserotonin receptor subtype (5-HT1F) in human brain: Interaction with aGTP-binding protein. J. Neurochem. 53(2):465-471, 1989.

Maniatis, T., Fritsch, E. F., and Sambrook, J. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1982.

Osborne, N. N. and Hamon, M.: Neuronal serotonin. Chichester: John Wileyand Sons, Inc., 1988.

Peroutka, S. J.: Serotonin receptor subtypes: Basic and clinicalaspects. New York: Wiley-Liss, Inc., 1991.

Peroutka, S. J. and Snyder, S. H.: Multiple serotonin receptors,differential binding of [³ H]5-hydroxytryptamine, [³ H]lysergic aciddiethylamide and [³ H]spiroperidol. Mol. Pharmacol. 16:687-699, 1979.

Pritchett, D. B., Bach, A. W. J., Wozny, M., Taleb, O., Dal Toso, R.,Shih, J. and Seeburg, P. H.: Structure and functional expression ofcloned rat serotonin 5-HT2 receptor. EMBO J. 7:4135-4140, 1988.

Rapport, M. M., Green, A. A. and Page, I. H.: Purification of the thesubstance which is responsible for vasoconstrictor activity of serum.Fed. Proc. 6:184, 1947.

Rapport, M. M.: Serum vasoconstrictor (serotonin) V. Presence ofcreatinine in the complex. A proposed structure of the vasoconstrictorprinciple. J. Biol. Chem. 180:961-969, 1949.

Sanders-Bush, E.: The Serotonin Receptors. Clifton, N.J.: Humana Press,1988.

Sleight, A. J., Pierce, P. A., Schmidt, A. W., Hekmatpanah, C. R. andPeroutka, S. J.: The clinical utility of serotonin receptor activeagents in neuropsychiatric disease. In: Serotonin receptor subtypes:Basic and clinical aspects. (ed. Peroutka, S. J.) New York:Wiley-Liss,Inc., pp 211-227, 1990.

Sumner, M. J. and Humphrey, P. P. A.: Sumatriptan (GR43175) inhibitscyclic-AMP accumulation in dog isolated saphenous vein. Br. J.Pharmacol. 99:219-220,

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 9    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1730 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (vii) IMMEDIATE SOURCE:    (A) LIBRARY: human lymphocyte genomic    (B) CLONE: hl16a    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 616..1713    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 616..1713    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCCTTTGTT60    ATTTTGTCATGCTTCAAGCCTAGGAAAAGCCTAAGCAAAACTCTTGGTGGGCTCTTTGTT120    ACATTCCAGCCTTTGAATAAGGGCACTGGCTCTATCAGCTTTGAATATATAACTCAACTA180    GTCAGTCAGTAGTACTGAAACAGTTGTTACGGAGGCCTGCGTTATTGAGATCGGGCCTGC240    CACACTTTTAAACTTTTTCTGACATGGACAAAGAGAAAAACCAATTCTATAATGGCAGAG300    ATTTCACTGAGTAACAAGCTAGAGTATCATTAAAAATTGTTGTATTTAACCTATATTTTA360    AGAAATGTTTTGGAAGTTACTGGCTTTTTTTACTGTTCTCATTAAATTTCTTAAATAAAA420    AGGAAAACTAAAACCTTCAATCTGAACCTCATTTTTTTAATCTATAGAATATTCTGGGTA480    AACATAACATACACTTTTTAAAAATTATTCTGAAAGGAAGAGAAAAGTTCTTGAAGCCTT540    CTCTGAACTGTTTTTTCTCTTCCCTTGTTACAGGTATCCATTTTTCAGCTATATTAATCT600    TTTAAAACAAAGAAAATGGATTTCTTAAATTCATCTGATCAAAACTTGACC651    MetAspPheLeuAsnSerSerAspGlnAsnLeuThr    1510    TCAGAGGAACTGTTAAACAGAATGCCATCCAAAATTCTGGTGTCCCTC699    SerGluGluLeuLeuAsnArgMetProSerLysIleLeuValSerLeu    152025    ACTCTGTCTGGGCTGGCACTGATGACAACAACTATCAACTCCCTTGTG747    ThrLeuSerGlyLeuAlaLeuMetThrThrThrIleAsnSerLeuVal    303540    ATCGCTGCAATTATTGTGACCCGGAAGCTGCACCATCCAGCCAATTAT795    IleAlaAlaIleIleValThrArgLysLeuHisHisProAlaAsnTyr    45505560    TTAATTTGTTCCCTTGCAGTCACAGATTTTCTTGTGGCTGTCCTGGTG843    LeuIleCysSerLeuAlaValThrAspPheLeuValAlaValLeuVal    657075    ATGCCCTTCAGCATTGTGTATATTGTGAGAGAGAGCTGGATTATGGGG891    MetProPheSerIleValTyrIleValArgGluSerTrpIleMetGly    808590    CAAGTGGTCTGTGACATTTGGCTGAGTGTTGACATTACCTGCTGCACG939    GlnValValCysAspIleTrpLeuSerValAspIleThrCysCysThr    95100105    TGCTCCATCTTGCATCTCTCAGCTATAGCTTTGGATCGGTATCGAGCA987    CysSerIleLeuHisLeuSerAlaIleAlaLeuAspArgTyrArgAla    110115120    ATCACAGATGCTGTTGAGTATGCCAGGAAAAGGACTCCAAAGCATGCT1035    IleThrAspAlaValGluTyrAlaArgLysArgThrProLysHisAla    125130135140    GGCATTATGATTACAATAGTTTGGATTATATCTGTTTTTATCTCTATG1083    GlyIleMetIleThrIleValTrpIleIleSerValPheIleSerMet    145150155    CCTCCTCTATTCTGGAGGCACCAAGGAACTAGCAGAGATGATGAATGC1131    ProProLeuPheTrpArgHisGlnGlyThrSerArgAspAspGluCys    160165170    ATCATCAAGCACGACCACATTGTTTCCACCATTTACTCAACATTTGGA1179    IleIleLysHisAspHisIleValSerThrIleTyrSerThrPheGly    175180185    GCTTTCTACATCCCACTGGCATTGATTTTGATCCTTTACTACAAAATA1227    AlaPheTyrIleProLeuAlaLeuIleLeuIleLeuTyrTyrLysIle    190195200    TATAGAGCAGCAAAGACATTATACCACAAGAGACAAGCAAGTAGGATT1275    TyrArgAlaAlaLysThrLeuTyrHisLysArgGlnAlaSerArgIle    205210215220    GCAAAGGAGGAGGTGAATGGCCAAGTCCTTTTGGAGAGTGGTGAGAAA1323    AlaLysGluGluValAsnGlyGlnValLeuLeuGluSerGlyGluLys    225230235    AGCACTAAATCAGTTTCCACATCCTATGTACTAGAAAAGTCTTTATCT1371    SerThrLysSerValSerThrSerTyrValLeuGluLysSerLeuSer    240245250    GACCCATCAACAGACTTTGATAAAATTCATAGCACAGTGAGAAGTCTC1419    AspProSerThrAspPheAspLysIleHisSerThrValArgSerLeu    255260265    AGGTCTGAATTCAAGCATGAGAAATCTTGGAGAAGGCAAAAGATCTCA1467    ArgSerGluPheLysHisGluLysSerTrpArgArgGlnLysIleSer    270275280    GGTACAAGAGAACGGAAAGCAGCCACTACCCTGGGATTAATCTTGGGT1515    GlyThrArgGluArgLysAlaAlaThrThrLeuGlyLeuIleLeuGly    285290295300    GCATTTGTAATATGTTGGCTTCCTTTTTTTGTAAAAGAATTAGTTGTT1563    AlaPheValIleCysTrpLeuProPhePheValLysGluLeuValVal    305310315    AATGTCTGTGACAAATGTAAAATTTCTGAAGAAATGTCCAATTTTTTG1611    AsnValCysAspLysCysLysIleSerGluGluMetSerAsnPheLeu    320325330    GCATGGCTTGGGTATCTCAATTCCCTTATAAATCCACTGATTTACACA1659    AlaTrpLeuGlyTyrLeuAsnSerLeuIleAsnProLeuIleTyrThr    335340345    ATCTTTAATGAAGACTTCAAGAAAGCATTCCAAAAGCTTGTGCGATGT1707    IlePheAsnGluAspPheLysLysAlaPheGlnLysLeuValArgCys    350355360    CGATGTTAGTTTTAAAAATGTTT1730    ArgCys    365    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 366 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAspPheLeuAsnSerSerAspGlnAsnLeuThrSerGluGluLeu    151015    LeuAsnArgMetProSerLysIleLeuValSerLeuThrLeuSerGly    202530    LeuAlaLeuMetThrThrThrIleAsnSerLeuValIleAlaAlaIle    354045    IleValThrArgLysLeuHisHisProAlaAsnTyrLeuIleCysSer    505560    LeuAlaValThrAspPheLeuValAlaValLeuValMetProPheSer    65707580    IleValTyrIleValArgGluSerTrpIleMetGlyGlnValValCys    859095    AspIleTrpLeuSerValAspIleThrCysCysThrCysSerIleLeu    100105110    HisLeuSerAlaIleAlaLeuAspArgTyrArgAlaIleThrAspAla    115120125    ValGluTyrAlaArgLysArgThrProLysHisAlaGlyIleMetIle    130135140    ThrIleValTrpIleIleSerValPheIleSerMetProProLeuPhe    145150155160    TrpArgHisGlnGlyThrSerArgAspAspGluCysIleIleLysHis    165170175    AspHisIleValSerThrIleTyrSerThrPheGlyAlaPheTyrIle    180185190    ProLeuAlaLeuIleLeuIleLeuTyrTyrLysIleTyrArgAlaAla    195200205    LysThrLeuTyrHisLysArgGlnAlaSerArgIleAlaLysGluGlu    210215220    ValAsnGlyGlnValLeuLeuGluSerGlyGluLysSerThrLysSer    225230235240    ValSerThrSerTyrValLeuGluLysSerLeuSerAspProSerThr    245250255    AspPheAspLysIleHisSerThrValArgSerLeuArgSerGluPhe    260265270    LysHisGluLysSerTrpArgArgGlnLysIleSerGlyThrArgGlu    275280285    ArgLysAlaAlaThrThrLeuGlyLeuIleLeuGlyAlaPheValIle    290295300    CysTrpLeuProPhePheValLysGluLeuValValAsnValCysAsp    305310315320    LysCysLysIleSerGluGluMetSerAsnPheLeuAlaTrpLeuGly    325330335    TyrLeuAsnSerLeuIleAsnProLeuIleTyrThrIlePheAsnGlu    340345350    AspPheLysLysAlaPheGlnLysLeuValArgCysArgCys    355360365    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 422 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (vii) IMMEDIATE SOURCE:    (B) CLONE: 5-HT1A    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    MetAspValLeuSerProGlyGlnGlyAsnAsnThrThrSerProPro    151015    AlaProPheGluThrGlyGlyAsnThrThrGlyIleSerAspValThr    202530    ValSerTyrGlnValIleThrSerLeuLeuLeuGlyThrLeuIlePhe    354045    CysAlaValLeuGlyAsnAlaCysValValAlaAlaIleAlaLeuGlu    505560    ArgSerLeuGlnAsnValAlaAsnTyrLeuIleGlySerLeuAlaVal    65707580    ThrAspLeuMetValSerValLeuValLeuProMetAlaAlaLeuTyr    859095    GlnValLeuAsnLysTrpThrLeuGlyGlnValThrCysAspLeuPhe    100105110    IleAlaLeuAspValLeuCysCysThrSerSerIleLeuHisLeuCys    115120125    AlaIleAlaLeuAspArgTyrTrpAlaIleThrAspProIleAspTyr    130135140    ValAsnLysArgThrProArgArgAlaAlaAlaLeuIleSerLeuThr    145150155160    TrpLeuIleGlyPheLeuIleSerIleProProMetLeuGlyTrpArg    165170175    ThrProGluAspArgSerAspProAspAlaCysThrIleSerLysAsp    180185190    HisGlyTyrThrIleTyrSerThrPheGlyAlaPheTyrIleProLeu    195200205    LeuLeuMetLeuValLeuTyrGlyArgIlePheArgAlaAlaArgPhe    210215220    ArgIleArgLysThrValLysLysValGluLysThrGlyAlaAspThr    225230235240    ArgHisGlyAlaSerProAlaProGlnProLysLysSerValAsnGly    245250255    GluSerGlySerArgAsnTrpArgLeuGlyValGluSerLysAlaGly    260265270    GlyAlaLeuCysAlaAsnGlyAlaValArgGlnGlyAspAspGlyAla    275280285    AlaLeuGluValIleGluValHisArgValGlyAsnSerLysGluHis    290295300    LeuProLeuProSerGluAlaGlyProThrProCysAlaProAlaSer    305310315320    PheGluArgLysAsnGluArgAsnAlaGluAlaLysArgLysMetAla    325330335    LeuAlaArgGluArgLysThrValLysThrLeuGlyIleIleMetGly    340345350    ThrPheIleLeuCysTrpLeuProPhePheIleValAlaLeuValLeu    355360365    ProPheCysGluSerSerCysHisMetProThrLeuLeuGlyAlaIle    370375380    IleAsnTrpLeuGlyTyrSerAsnSerLeuLeuAsnProValIleTyr    385390395400    AlaTyrPheAsnLysAspPheGlnAsnAlaPheLysLysIleIleLys    405410415    CysLeuPheCysArgGln    420    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 460 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (vii) IMMEDIATE SOURCE:    (B) CLONE: 5-HT1C    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetValAsnLeuGlyAsnAlaValArgSerLeuLeuMetHisLeuIle    151015    GlyLeuLeuValTrpGlnPheAspIleSerIleSerProValAlaAla    202530    IleValThrAspThrPheAsnSerSerAspGlyGlyArgLeuPheGln    354045    PheProAspGlyValGlnAsnTrpProAlaLeuSerIleValValIle    505560    IleIleMetThrIleGlyGlyAsnIleLeuValIleMetAlaValSer    65707580    MetGluLysLysLeuHisAsnAlaThrAsnTyrPheLeuMetSerLeu    859095    AlaIleAlaAspMetLeuValGlyLeuLeuValMetProLeuSerLeu    100105110    LeuAlaIleLeuTyrAspTyrValTrpProLeuProArgTyrLeuCys    115120125    ProValTrpIleSerLeuAspValLeuPheSerThrAlaSerIleMet    130135140    HisLeuCysAlaIleSerLeuAspArgTyrValAlaIleArgAsnPro    145150155160    IleGluHisSerArgPheAsnSerArgThrLysAlaIleMetLysIle    165170175    AlaIleValTrpAlaIleSerIleGlyValSerValProIleProVal    180185190    IleGlyLeuArgAspGluSerLysValPheValAsnAsnThrThrCys    195200205    ValLeuAsnAspProAsnPheValLeuIleGlySerPheValAlaPhe    210215220    PheIleProLeuThrIleMetValIleThrTyrPheLeuThrIleTyr    225230235240    ValLeuArgArgGlnThrLeuMetLeuLeuArgGlyHisThrGluGlu    245250255    GluLeuAlaAsnMetSerLeuAsnPheLeuAsnCysCysCysLysLys    260265270    AsnGlyGlyGluGluGluAsnAlaProAsnProAsnProAspGlnLys    275280285    ProArgArgLysLysLysGluLysArgProArgGlyThrMetGlnAla    290295300    IleAsnAsnGluLysLysAlaSerLysValLeuGlyIleValPhePhe    305310315320    ValPheLeuIleMetTrpCysProPhePheIleThrAsnIleLeuSer    325330335    ValLeuCysGlyLysAlaCysAsnGlnLysLeuMetGluLysLeuLeu    340345350    AsnValPheValTrpIleGlyTyrValCysSerGlyIleAsnProLeu    355360365    ValTyrThrLeuPheAsnLysIleTyrArgArgAlaPheSerLysTyr    370375380    LeuArgCysAspTyrLysProAspLysLysProProValArgGlnIle    385390395400    ProArgValAlaAlaThrAlaLeuSerGlyArgGluLeuAsnValAsn    405410415    IleTyrArgHisThrAsnGluArgValAlaArgLysAlaAsnAspPro    420425430    GluProGlyIleGluMetGlnValGluAsnLeuGluLeuProValAsn    435440445    ProSerAsnValValSerGluArgIleSerSerVal    450455460    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 376 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (vii) IMMEDIATE SOURCE:    (B) CLONE: 5-HT1DA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    MetSerProLeuAsnGlnSerAlaGluGlyLeuProGlnGluAlaSer    151015    AsnArgSerLeuAsnAlaThrGluThrSerGluAlaTrpAspProArg    202530    ThrLeuGlnAlaLeuLysIleSerLeuProValLeuLeuSerValIle    354045    ThrLeuAlaThrValLeuSerAsnAlaPheValLeuThrThrIleLeu    505560    LeuThrArgLysLeuHisThrProAlaAsnTyrLeuIleGlySerLeu    65707580    AlaThrThrAspLeuLeuValSerIleLeuValMetProIleSerMet    859095    AlaTyrThrIleThrHisThrTrpAsnPheGlyGlnIleLeuCysAsp    100105110    IleTrpLeuSerSerAspIleThrCysCysThrAlaSerIleLeuHis    115120125    LeuCysValIleAlaLeuAspArgTyrTrpAlaIleThrAspAlaLeu    130135140    GluTyrSerLysArgArgThrAlaGlyHisAlaAlaThrMetIleAla    145150155160    IleValTrpAlaIleSerIleCysIleSerIleProProLeuPheTrp    165170175    ArgGlnGluLysAlaGlnGluGluMetSerAspCysLeuValAsnThr    180185190    SerGlnIleSerTyrThrIleTyrSerThrCysGlyAlaPheTyrIle    195200205    ProSerValLeuLeuIleIleLeuTyrGlyArgIleTyrArgAlaAla    210215220    ArgAsnArgIleLeuAsnProProSerLeuSerGlyLysArgPheThr    225230235240    ThrAlaHisLeuIleThrGlySerAlaGlySerValCysSerLeuAsn    245250255    SerSerLeuHisGluGlyHisSerHisSerAlaGlySerProLeuPhe    260265270    PheAsnHisValLysIleLysLeuAlaAspSerAlaLeuGluArgLys    275280285    ArgIleSerAlaAlaArgGluArgLysAlaThrLysIleLeuGlyIle    290295300    IleLeuGlyAlaPheIleIleCysTrpLeuProPhePheValValSer    305310315320    LeuValLeuProIleCysArgAspSerCysTrpIleHisProGlyLeu    325330335    PheAspPhePheThrTrpLeuGlyTyrLeuAsnSerLeuIleAsnPro    340345350    IleIleTyrThrValPheAsnGluGluPheArgGlnAlaPheGlnLys    355360365    IleValProPheArgLysAlaSer    370375    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 390 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (vii) IMMEDIATE SOURCE:    (B) CLONE: 5-HT1DB    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetGluGluProGlyAlaGlnCysAlaProProAlaProAlaGlySer    151015    GluThrTrpValProGlnAlaAsnLeuSerSerAlaProSerGlnAsn    202530    CysSerAlaLysAspTyrIleTyrGlnAspSerIleSerLeuProTrp    354045    LysValLeuLeuValMetLeuLeuAlaLeuIleThrLeuAlaThrThr    505560    LeuSerAsnAlaPheValIleAlaThrValTyrArgThrArgLysLeu    65707580    HisThrProAlaAsnTyrLeuIleAlaSerLeuAspValThrAspLeu    859095    LeuValSerIleLeuValIleProIleSerThrMetTyrThrValThr    100105110    AspArgTrpThrLeuSerGlnValValCysAspPheTrpLeuSerSer    115120125    AspIleThrCysCysThrAlaSerIleLeuHisLeuCysValIleAla    130135140    LeuAspArgTyrTrpAlaIleThrAspAlaValGluTyrSerAlaLys    145150155160    ArgThrProLysArgAlaAlaValMetIleAlaLeuValTrpValPhe    165170175    SerIleSerIleSerLeuProProPhePheTrpArgGlnAlaLysAla    180185190    GluGluGluValSerGluCysValValAsnThrAspHisIleLeuTyr    195200205    ThrValTyrSerThrValGlyAlaPheTyrPheProThrLeuLeuLeu    210215220    IleAlaLeuTyrGlyArgIleTyrValGluAlaArgSerArgIleLeu    225230235240    LysGlnThrProAsnArgThrGlyLysArgLeuThrArgAlaGlnLeu    245250255    IleThrAspSerProGlySerThrSerSerValThrSerIleAsnSer    260265270    ArgValProAspValProSerGluSerGlySerProValTyrValAsn    275280285    GlnValLysValArgValSerAspAlaLeuLeuGluLysLysLysLeu    290295300    MetAlaAlaArgGluArgLysAlaThrLysThrLeuGlyIleIleLeu    305310315320    GlyAlaPheIleValCysTrpLeuProPhePheIleIleSerLeuVal    325330335    MetProIleCysLysAspAlaCysTrpPheHisLeuAlaIlePheAsp    340345350    PhePheThrTrpLeuGlyTyrLeuAsnSerLeuIleAsnProIleIle    355360365    TyrThrMetSerAsnGluAspPheLysGlnAlaPheHisLysLeuIle    370375380    ArgPheLysCysThrSer    385390    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 366 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (vii) IMMEDIATE SOURCE:    (B) CLONE: 5-HT1F    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    MetAspPheLeuAsnSerSerAspGlnAsnLeuThrSerGluGluLeu    151015    LeuAsnArgMetProSerLysIleLeuValSerLeuThrLeuSerGly    202530    LeuAlaLeuMetThrThrThrIleAsnSerLeuValIleAlaAlaIle    354045    IleValThrArgLysLeuHisHisProAlaAsnTyrLeuIleCysSer    505560    LeuAlaValThrAspPheLeuValAlaValLeuValMetProPheSer    65707580    IleValTyrIleValArgGluSerTrpIleMetGlyGlnValValCys    859095    AspIleTrpLeuSerValAspIleThrCysCysThrCysSerIleLeu    100105110    HisLeuSerAlaIleAlaLeuAspArgTyrArgAlaIleThrAspAla    115120125    ValGluTyrAlaArgLysArgThrProLysHisAlaGlyIleMetIle    130135140    ThrIleValTrpIleIleSerValPheIleSerMetProProLeuPhe    145150155160    TrpArgHisGlnGlyThrSerArgAspAspGluCysIleIleLysHis    165170175    AspHisIleValSerThrIleTyrSerThrPheGlyAlaPheTyrIle    180185190    ProLeuAlaLeuIleLeuIleLeuTyrTyrLysIleTyrArgAlaAla    195200205    LysThrLeuTyrHisLysArgGlnAlaSerArgIleAlaLysGluGlu    210215220    ValAsnGlyGlnValLeuLeuGluSerGlyGluLysSerThrLysSer    225230235240    ValSerThrSerTyrValLeuGluLysSerLeuSerAspProSerThr    245250255    AspPheAspLysIleHisSerThrValArgSerLeuArgSerGluPhe    260265270    LysHisGluLysSerTrpArgArgGlnLysIleSerGlyThrArgGlu    275280285    ArgLysAlaAlaThrThrLeuGlyLeuIleLeuGlyAlaPheValIle    290295300    CysTrpLeuProPhePheValLysGluLeuValValAsnValCysAsp    305310315320    LysCysLysIleSerGluGluMetSerAsnPheLeuAlaTrpLeuGly    325330335    TyrLeuAsnSerLeuIleAsnProLeuIleTyrThrIlePheAsnGlu    340345350    AspPheLysLysAlaPheGlnLysLeuValArgCysArgCys    355360365    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 471 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (vii) IMMEDIATE SOURCE:    (B) CLONE: 5-HT2    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    MetAspIleLeuCysGluGluAsnThrSerLeuSerSerThrThrAsn    151015    SerLeuMetGlnLeuAsnAspAspThrArgLeuTyrSerAsnAspPhe    202530    AsnSerGlyGluAlaAsnThrSerAspAlaPheAsnTrpThrValAsp    354045    SerGluAsnArgThrAsnLeuSerCysGluGlyCysLeuSerProSer    505560    CysLeuSerLeuLeuHisLeuGlnGluLysAsnTrpSerAlaLeuLeu    65707580    ThrAlaValValIleIleLeuThrIleAlaGlyAsnIleLeuValIle    859095    MetAlaValSerLeuGluLysLysLeuGlnAsnAlaThrAsnTyrPhe    100105110    LeuMetSerLeuAlaIleAlaAspMetLeuLeuGlyPheLeuValMet    115120125    ProValSerMetLeuThrIleLeuTyrGlyTyrArgTrpProLeuPro    130135140    SerLysLeuCysAlaValTrpIleTyrLeuAspValLeuPheSerThr    145150155160    AlaSerIleMetHisLeuCysAlaIleSerLeuAspArgTyrValAla    165170175    IleGlnAsnProIleHisHisSerArgPheAsnSerArgThrLysAla    180185190    PheLeuLysIleIleAlaValTrpThrIleSerValGlyIleSerMet    195200205    ProIleProValPheGlyLeuGlnAspAspSerLysValPheLysGlu    210215220    GlySerCysLeuLeuAlaAspAspAsnPheValLeuIleGlySerPhe    225230235240    ValSerPhePheIleProLeuThrIleMetValIleThrTyrPheLeu    245250255    ThrIleLysSerLeuGlnLysGluAlaThrLeuCysValSerAspLeu    260265270    GlyThrArgAlaLysLeuAlaSerPheSerPheLeuProGlnSerSer    275280285    LeuSerSerGluLysLeuPheGlnArgSerIleHisArgGluProGly    290295300    SerTyrThrGlyArgArgThrMetGlnSerIleSerAsnGluGlnLys    305310315320    AlaCysLysValLeuGlyIleValPhePheLeuPheValValMetTrp    325330335    CysProPhePheIleThrAsnIleMetAlaValIleCysLysGluSer    340345350    CysAsnGluAspValIleGlyAlaLeuLeuAsnValPheValTrpIle    355360365    GlyTyrLeuSerSerAlaValAsnProLeuValTyrThrLeuPheAsn    370375380    LysThrTyrArgSerAlaPheSerArgTyrIleGlnCysGlnTyrLys    385390395400    GluAsnLysLysProLeuGlnLeuIleLeuValAsnThrIleProAla    405410415    LeuAlaTyrLysSerSerGlnLeuGlnMetGlyGlnLysLysAsnSer    420425430    LysGlnAspAlaLysThrThrAspAsnAspCysSerMetValAlaLeu    435440445    GlyLysGlnHisSerGluGluAlaSerLysAspAsnSerAspGlyVal    450455460    AsnGluLysValSerCysVal    465470    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 45 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vi) ORIGINAL SOURCE:    (A) ORGANISM: ANTISENSE OLIGO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    TCTCACCACTCTCCAAAAGGACTTGGCCATTCACCTCCTCCTTTG45    __________________________________________________________________________

What is claimed is:
 1. A process for identifying a chemical compound which specifically binds to a human 5-H_(1F) receptor, which comprises contacting a plurality of nonneuronal cells expressing on their cell surface the human 5-HT_(1F) receptor having the amino acid sequence shown in Seq. I.D. No. 2, with the chemical compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the receptor.
 2. A process for identifying a chemical compound which specifically binds to a human 5-HT_(1F) receptor, which comprises contacting a membrane fraction from a cell extract of nonneuronal cells expressing on their cell surface the human 5-HT_(1F) receptor having the amino acid sequence shown in Seq. I.D. No. 2, with the chemical compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the human 5-HT_(1F) receptor.
 3. A process involving competitive binding for identifying a chemical compound which specifically binds to a human 5-HT_(1F) receptor, which comprises separately contacting nonneuronal cells expressing on their cell surface the human 5-HT_(1F) receptor having the amino acid sequence shown in Seq. I.D. No. 2, with both the chemical compound and a second chemical compound known to bind to the human 5-HT_(1F) receptor, and with only the second chemical compound under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the human 5-HT_(1F) receptor, a decrease in the binding of the second chemical compound to the human 5-HT_(1F) receptor in the presence of the chemical compound indicating that the chemical compound binds to the human 5-HT_(1F) receptor.
 4. A process involving competitive binding for identifying a chemical compound which specifically binds to a human 5-HT_(1F) receptor, which comprises separately contacting a membrane fraction from a cell extract of nonneuronal cells expressing on their cell surface the human 5-HT_(1F) receptor having the amino acid sequence shown in Seq. I.D. No. 2, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the human 5-HT_(1F) receptor, a decrease in binding of the second chemical compound to the human 5-HT_(1F) receptor in the presence of the chemical compound indicating that the chemical compound binds to the human 5-HT_(1F) receptor.
 5. A process for determining whether a chemical compound specifically binds to and activates a human 5-HT_(1F) receptor, which comprises contacting nonneuronal cells producing cAMP and expressing on their cell surface the human 5-HT_(1F) receptor having the amino acid sequence shown in Seq. I.D. No. 2, with the chemical compound under conditions suitable for activation of the human 5-HT_(1F) receptor, and measuring cAMP in the presence and in the absence of the chemical compound, a decrease in cAMP in the presence of the compound indicating that the compound activates the human 5-HT_(1F) receptor.
 6. A process for determining whether a chemical compound specifically binds to and activates a human 5-HT_(1F) receptor, which comprises contacting a membrane fraction from a cell extract of nonneuronal cells producing cAMP and expressing on their cell surface the human 5-HT_(1F) receptor having the amino acid sequence shown in Seq. I.D. No. 2, with the chemical compound under conditions suitable for activation of the human 5-HT_(1F) receptor, and measuring cAMP in the presence and in the absence of the chemical compound, a decrease in cAMP formation in the presence of the chemical compound indicating that the chemical compound activates the human 5-HT_(1F) receptor.
 7. A process for determining whether a chemical compound specifically binds to and inhibits activation of a human 5-HT_(1F) receptor, which comprises separately contacting nonneuronal cells producing cAMP and expressing on their cell surface the human 5-HT_(1F) receptor having the amino acid sequence shown in Seq. I.D. No. 2, with both the chemical compound and a second chemical compound known to activate the human 5-HT_(1F) receptor and with only the second chemical compound, under conditions suitable for activation of the human 5-HT_(1F) receptor, measuring cAMP in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller decrease in cAMP in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the human 5-HT_(1F) receptor.
 8. A process for determining whether a chemical compound specifically binds to and inhibits activation of a human 5-HT_(1F) receptor, which comprises separately contacting a membrane fraction from a cell extract of nonneuronal cells producing cAMP and expressing on their cell surface the human 5-HT_(1F) receptor having the amino acid sequence shown in Seq. I.D. No. 2, with both the chemical compound and a second chemical compound known to activate the human 5-HT_(1F) receptor and with only the second chemical compound, under conditions suitable for activation of the human 5-HT_(1F) receptor, measuring cAMP in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller decrease in cAMP in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the human 5-HT_(1F) receptor.
 9. The process of any one of claims 1, 2, 3, 4, 5, 6, 7 or 8, wherein the nonneuronal cell is a mammalian cell.
 10. The process of claim 9, wherein the nonneuronal mammalian cell is an NIH-3T3 cell.
 11. The process of claim 9, wherein the nonneuronal mammalian cell is an Ltk- cell. 